Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
  • C08B37/0078—Degradation products
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• Coagulation is a physiological pathway involved in maintaining normal blood hemostasis in mammals. Under conditions in which a vascular injury occurs, the coagulation pathway is stimulated to form a blood clot to prevent the loss of blood. Immediately after the vascular injury occurs, blood platelets begin to aggregate at the site of injury forming a physical plug to stop the leakage. In addition, the injured vessel undergoes vasoconstriction to reduce the blood flow to the area and fibrin begins to aggregate forming an insoluble network or clot, which covers the ruptured area.
• thrombotic tendencies which are often manifested as heart attacks, strokes, deep vein thrombosis, and acute coronary syndromes such as myocardial infarcts, and unstable angina.
• an embolism can break off from a thrombus and result in a pulmonary embolism or cerebral vascular embolism including stroke or transient ischemia attack.
• Current therapies for treating disorders associated with imbalances in the coagulation pathway involve many risks and must be carefully controlled.
• Heparin and low molecular weight heparins are potent modulators of hemostasis.
• Heparin, a highly sulfated heparin-like glycosaminoglycan (HLGAG) produced by mast cells is a widely used clinical anticoagulant, and is one of the first biopolymeric drugs and one of the few carbohydrate drugs.
• Heparin and molecules derived from it are potent anticoagulants that are used in a variety of clinical situations, especially for thromboembolic disorders including the prophylaxis and treatment of deep venous thrombosis and pulmonary embolism, arterial thromboses, and acute coronary syndromes like myocardial infarction and unstable angina.
• Heparin and LMWHs interact with multiple components of the coagulation cascade to inhibit the clotting process. Heparin primarily elicits its effect through two mechanisms, both of which involve binding of antithrombin III (AT-III) to a specific pentasaccharide sequence, HNAC/S,6SGHNS,3S,6SI2SHNS,6S contained within the polymer.
• AT-III antithrombin III
• AT-III binding to the pentasaccharide induces a conformational change in the protein that mediates its inhibition of factor Xa.
• thrombin factor Ha
• thrombin also binds to heparin at a site proximate to the pentasaccharide/ AT-III binding site. Formation of a ternary complex between AT-III, thrombin and heparin results in inactivation of thrombin.
• heparin's anti-IIa activity is size-dependent, in addition to the pentasaccharide unit responsible for anti-Xa activity for the efficient formation of an AT- III, thrombin, and heparin ternary complex. Heparin also mediates the release of tissue factor pathway inhibitor (TFPI) from endothelial cells.
• TFPI tissue factor pathway inhibitor
• TFPI a heparin cofactor
• TFPI is a serine protease that directly binds to and inhibits factor X.
• TFPI is a potent antithrombotic, particularly when co-administered with heparin.
• heparin Although heparin is highly efficacious in a variety of clinical situations and has the potential to be used in many others, the side effects associated with heparin therapy are many and varied. Anti-coagulation has been the primary clinical application for unfractionated heparin (UFH) for over 65 years. Due to its erratic intravenous pharmacokinetics and lack of subcutaneous bioavailability, UFH has been administered by intravenous injection instead. Additionally, the application of UFH as an anticoagulant has been hampered by the many side effects associated with non-specific plasma protein binding with UFH.
• UHF unfractionated heparin
• LMWH low molecular weight heparin
• LMWH provide a more predictable pharmacological action, reduced side effects, and better bioavailability than UFH. Since the commercially available LMWH preparations are not fully neutralized by protamine, an unexpected reaction could have extremely adverse effects; the anti-Xa activity of enoxaparin and other LMWH are neutralizable only to an extent of about 40% with ⁇ 2 mg Protamine/100 IU anti-Xa LMWH. The anti-IIa activity is neutralizable only to an extent of about 60% with ⁇ 2 mg Protamine/100 IU anti-Xa LMWH. (On the other hand, the anti-Xa and anti-IIa activity of UFH is neutralizable almost completely (>90%) with ⁇ 2 mg Protamine sulfate/100 IU anti-Xa UFH.)
• compositions of these polysaccharides are heterogeneous in length and composition. As such, only a portion of a typical preparation possesses anticoagulant activity.
• the majority of the polysaccharide chains in a pharmaceutical preparation of heparin or LMWH are inactive, at worst, these chains interact nonspecifically with plasma proteins to elicit the side effects associated with heparin therapy. Therefore, it is important to develop novel LMWHs that retain the anticoagulant activity and other desired activities of UFH but have reduced side effects.
• LMWHs essentially due to their reduced chains sizes and dispersity, display markedly less non-specific plasma protein binding.
• LMWHs that are currently clinically available also possess reduced anti-IIa activity as compared to UFH. Because of this decreased activity, a larger dose of LMWH is required (compared to UFH) in order to achieve a similar anti-Xa and anti-IIa activity, and the standard tests for UFH activity, activated partial thromboplastin time (aPTT) or activated clotting time (ACT), are not useful as they rely primarily on anti-IIa activity for a readout.
• aPTT activated partial thromboplastin time
• ACT activated clotting time
• the most widely used test for monitoring LMWH levels is an anti-Xa activity test, which depends on the subject having sufficient levels of antithrombin III (ATIII), which is not always the case.
• the invention is based, in part, on the development of preparations of LMWHs having, e.g., designed to have, improved properties, e.g., properties that provide a clinical advantage.
• improved properties include, by way of example, one or more of: reversibility in response to protamine sulfate; predictable or otherwise improved pharmacokinetics; improved anti-IIa activity, as compared, e.g., to enoxaparin; a relatively constant anti-Xa activity to anti-IIa activity ratio over a period of about 30 to 180 minutes; monitorable activity levels; subcutaneous bioavailability; and reduced likelihood of causing heparin induced thrombocytopenia (HIT).
• HIT heparin induced thrombocytopenia
• LMWHs disclosed herein can also have structural characteristics that distinguish them from other commercially available LMWHs.
• a LMWH preparation provided herein can have one or more of the following characteristics: substantially undetectable linkage region; an increased amount of 3-0 sulfates as compared to commercially available LMWH preparations; a subset of the chains have an unsulfated ⁇ U at the non-reducing end; a subset of the chains, e.g., a majority, e.g., substantially all of the chains, have an N-acetylated hexosamine at the reducing end; a ratio of ⁇ UHNA C ,6SGHNS,3S,6S to ⁇ U 2S H NS ,6 S IH N AC,6 S GH NS ,3 S ,6 S of about 1 :1 to 4:1 (e.g., about 1 :1, 2:1, 3:1, 4:1), and substantially no modified reducing end structures.
• the invention includes LMWH preparations having one or more
• a LMWH composition having: a weight average molecular weight of about 5000 to 9000 Da, e.g., about 5000 to 8300 Da, e.g., about 5500 to 8000 Da, e.g., about 5700 to 7900 Da, e.g., about 5800 to 6800 Da; and an anti-IIa activity of about 50 to 300, e.g., about 70 to 280, e.g., about 90 to 250 IU/mg, e.g., about 100 to 250 IU/mg, e.g., about 100 to 140 IU/mg, 150 to 200 IU/mg, about 130 to 190 IU/mg, e.g., about 155 to 195 IU/mg.
• the invention features, a LMWH composition having: a weight average molecular weight of about 5000 to 9000 Da, e.g., about 5000 to 8300 Da, e.g., about 5000 to 8000 Da, about 5500 to 8000 Da, e.g., about 5700 to 7900 Da, e.g., about 5800 to 6800 Da; and anti-IIa activity that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% neutralizable with protamine, e.g., as measured by activated partial thromboplastin time (ACT) or activated partial thromboplastin time (aPTT).
• ACT activated partial thromboplastin time
• aPTT activated partial thromboplastin time
• the anti-IIa activity of the LMWH is neutralized by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% within 5, 10, 15, 30 minutes after protamine administration.
• the invention features, a LMWH composition having: a weight average molecular weight of about 5000 to 9000 Da, e.g., about 5000 to 8300 Da, e.g., about 5500 to 8000 Da, e.g., about 5700 to 7900 Da, e.g., about 5800 to 6800 Da; and
• ⁇ UH NA c,6sGH N s,3s,6s is 5 to 15%, e.g., 7 to 14%, e.g. , 9 to 12%, of the composition, e.g., as measured by mole%.
• the ⁇ UHNA C ,6 S GHN S ,3 S ,6 S at the non- reducing end of the molecule of about 5 to 15%, e.g., 7 to 14%, e.g., 9 to 12%, of the chains in the composition, e.g., as measured by mole%.
• the invention features, a LMWH composition having: an average chain length of about 9 to 18 disaccharides or 8 to 18 disaccharides, e.g., about 9 to 16 or 8 to 16 disaccharides; and
• ⁇ UH NA c,6sGH N s,3s,6s is 5 to 15%, e.g., 7 to 14%, e.g. , 9 to 12%, of the composition, e.g., as measured by mole%.
• the ⁇ UHNAc,6sGH N s,3 S ,6 S. at the non- reducing end of the molecule of about 5tol5%, e.g., 7 to 14%, e.g., 9 to 12%, of the chains in the composition, e.g., as measured by mole%.
• the invention features, a LMWH composition having: a weight average molecular weight of 5000 to 9000 Da, e.g., about 5000 to 8300
• Da e.g., about 5500 to 8000 Da, e.g., about 5700 to 7900 Da, e.g., about 5800 to 6800
• an anti Xa to anti-IIa ratio of 3 : 1 or less e.g., 2:1, e.g., 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1 :1, 1 :1 or 0.5:1.
• the anti-Xa to anti-IIa ratio remains relatively constant over the course of an administration of the LMWH preparation, e.g., the anti-Xa to anti-IIa ratio varies no more than about + 1.5, ⁇ 1, ⁇ 0.5, or +0.2, over a period of about 30, 60, 120, 180, 240, 300 minutes.
• the ratio measured at a second time e.g., 30, 60, 120, 180, 240, 300 minutes
• the invention features, a LMWH composition having: optionally, a weight average molecular weight of about 5000 to 9000 Da, e.g., about 5000 to 8300 Da, e.g., about 5500 to 8000 Da, e.g., about 5700 to 7900 Da, e.g., about 5800 to 6800 Da; and when analyzed by digestion with heparinase I, heparinase II and heparinase III and capillary electrophoresis, each of peaks 1-14 of Table 1OA is present.
• the amount of each peak in the LMWH composition, as analyzed by digestion with heparinase I, heparinase II and heparinase III and capillary electrophoresis is about that found in Table 1OA, the amount of each peak is within a range provided in Table 1OA; the amount of peaks 10 and 11 are within a range provided in Table 1OA.
• the invention features, a LMWH composition having: optionally, a weight average molecular weight of about 5000 to 9000 Da, e.g., about 5000 to 8300 Da, e.g., about 5500 to 8000 Da, e.g., about 5700 to 7900 Da, e.g., about 5800 to 6800 Da; and when analyzed by 2D nuclear magnetic resonance (NMR) protons for each of the structures of Table 1 IA are present.
• NMR nuclear magnetic resonance
• the amount of each of the structures in the LMWH composition, as analyzed by 2D NMR is about that found in Table 1 IA.
• the invention features a LMWH composition having one or more of the following characteristics: the composition has substantially no (e.g., at least 85%, 90%, 95% or more of the chains do not have) modified reducing end structures; at least 60%, 70%, 80%, 85%, 90%, 95%, 99% of the chains of the composition have H NAC at the reducing end; less than 90%, 95%, 98%, 99%, preferably none of the chains of the composition have a sulfated ⁇ U at the non-reducing end; there is substantially no linkage region (e.g., less than 0.1% linkage region) present in the composition; the composition has more chains with 3-0 sulfates than commercially available LMWHs, e.g., enoxaparin or dalteparin; and the ratio of ⁇ UH NA c,6sGH
• the composition has two, three, four, five or all of these characteristics.
• the invention features a LMWH composition having the following structure:
• R is H or SO 3 X
• Rl is SO 3 X or COCH 3 ;
• X is a monovalent or divalent cation
• n 2-50, e.g., 2-40
• the composition preferably has an average value for n of 9-16, 8-16 or 8-15.
• the LMWH composition has the following structure:
• R is H or SO 3 X
• Rl is SO 3 X or COCH 3 ;
• X is a monovalent or divalent cation
• n 2-50, e.g., 2-40
• the composition preferably has an average value for n of 9-16, 8-16 or 8-15.
• the invention features, a LMWH composition having the following structure:
• X is Na or Ca, R is H or SO 3 Na;
• the LMWH composition has the following structure:
• X is Na or Ca, R is H or SO 3 Na;
• This composition can occur as an intermediate in the production of a LMWH, e.g., as the product of enzymatic digestion of the fast moving fraction (as discussed herein).
• the invention features, a LMWH composition having the following structure:
• X is Na or Ca
• R is H or SO 3 Na
• Rl is SO 3 Na or COCH 3
• n 2-50, e.g., 2-40
• the composition preferably has an average value for n of 8 to 15, e.g., 10 to 15, or 9 to 16, e.g., 11 to 16.
• the LMWH composition has the following structure:
• X is Na or Ca, R is H or SO 3 Na;
• This composition can occur as an intermediate in the production of a LMWH, e.g., as the product of precipitations to provide a fast moving fraction (as discussed herein).
• any of the LMWHs described herein, e.g., described above, can have other properties.
• one of the above described compositions can further have one or more of functional or structural properties set out below.
• the LMWH composition has an anti-Xa activity of about 100 to 400 IU/mg, e.g., about 120 to 380 IU/mg, e.g., about 150 to 350 IU/mg, e.g., about 170 to 330 IU/mg, e.g., about 180 to 300 IU/mg, e.g., about 150 to 200 IU/mg, 200 to 300 IU/mg, 130 to 220 IU/mg, 225 to 274 IU/mg.
• the LMWH composition has an anti-Xa activity that is at least 50%, 60%, 70%, 80%, 85%,90%, 95%, 99%, 100% neutralizable, e.g., as measured by anti-Xa activity, ACT or aPTT.
• the anti-Xa activity is neutralized by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% within 5, 10, 15 minutes after protamine administration.
• the anti-Xa activity can be neutralized by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% within 5, 10, 15, 30 minutes after protamine administration at a dose of about 1, 2, 3 mg of the LMWH composition per 100 anti-Xa IU of plasma.
• the LMWH composition has one or more of the following properties: the activity of the composition can be monitored by aPTT and/or ACT; the polydispersity of the composition is less than 1.6, e.g., the polydispersity is about 1.6 to 1.1, e.g., 1.5 to 1.1, e.g., 1.4 to 1.1, e.g., 1.3 to 1.1, e.g., 1.2 to 1.1; less than 70%, 60%, 50%, 45%, 40%, 35%, 30% of the chains present in the composition have a molecular weight greater than 7500 or 8000 Da; less than 40%, 35%, 30%, 25% of the chains present in the composition have a molecular weight less than 5500 or 5000 Da; the composition comprises a mixture of ⁇ U and I/G structures at the non-reducing ends of the chains; and fewer chains in the composition have PF4 binding sites than enoxaparin, dalteparin, UFH.
• about 15%, 20%, 25%, 30%, 35%, 45%, 50% of the chains in the LMWH composition have a ⁇ U at the non-reducing end.
• about 15% to 50%, e.g., 15% to 35% of the chains, e.g., 20% to 35% of the chains in the composition have a ⁇ U at the non-reducing end.
• the LMWH composition has a higher degree of sulfation than enoxaparin or dalteparin. In one embodiment, the LMWH composition has more trisulfated disaccharides present in the composition than enoxaparin or dalteparin, e.g., the LMWH composition has about 50 to 65% trisulfated disaccharides, e.g., 55 to 60%, 55 to 58%, 57 to 60% trisulfated disaccharides, as determined by mole%.
• the composition comprises a higher level of ⁇ UH NAC,6S GH NS,3S,6S than enoxaparin, daltaparin and/or UFH, e.g., comprises about 5 to 15 mole%, e.g., 7 to 14 mole%, e.g. , 9 to 12mole%.
• the LMWH composition has a calcium content less than 3%, 2.5%, 2%, 1.5%, 1.0%, and/or a sodium content less than 30%, 25%, 20%, 15%, 10%.
• the LMWH composition comprises: less than 1000 ng/mg, 750 ng/mg, 500 ng/mg, 250 ng/mg of a heparinase enzyme, e.g., a heparinase enzyme described herein; less than 1.0%, 0.5%, 0.3% w/w methanol; less than 1.0%, 0.5%, 0.3%, 0.1% w/w ethanol; less than 2.0%, 1.75%, 1.25%, 1.0%, 0.5%, 0.3%, 0.15% chloride; less than 15%, 10%, 5%, 2.5% water by weight; less than 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300 ppm of free sulf
• the LMWH composition provides increased TFPI release as compared to enoxaparin. In one embodiment, the LMWH provides at least a 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 fold increase in TFPI release as compared to enoxaparin.
• the LMWH composition has an intravenous half life of about 30 minutes to 3 hours, e.g., about 1 to 2 hours. In one embodiment, the LMWH composition has a subcutaneous half life of about 30 minutes to 3.0 or 3.5 hours, e.g., about 1.5 to 2.5 hours, e.g., about 2 hours.
• the LMWH composition has one or more of the following characteristics: the composition has substantially no (e.g., at least 85%, 90%, 95% or more of the chains do not have) modified reducing end structures; at least 60%, 70%, 80%, 85%, 90%, 95%, 99% of the chains of the composition have H NAC at the reducing end; less than 90%, 95%, 98%, 99%, preferably none of the chains of the composition have a sulfated ⁇ U at the non-reducing end; there is substantially no linkage region (e.g., less than 0.1% linkage region) present in the composition; the composition has more chains with 3-0 sulfates than commercially available LMWHs, e.g., enoxaparin or dalteparin; and the ratio of ⁇ UH N Ac,6sGH NS ,3s,6S to ⁇ U2SH N S,6
• the invention features, a LMWH composition having the following properties: a weight average molecular weight of about 5000 to 9000 Da; anti-IIa activity of about 50 to 300 IU/mg; anti-IIa activity that is at least 50% neutralizable with protamine, e.g., as measured by ACT or aPTT;
• ⁇ UH NAC,6S GH NSI3SI6S is 5 to 15% of the composition, preferably ⁇ UH NAc,6 sGH NSi3Si6S at the non-reducing end_of about 5 to 15% of the composition; an average chain length of about 9 to 16 disaccharides; an anti Xa to anti-IIa ratio of 3 : 1 or less; the anti-Xa to anti-IIa ratio remains relatively constant over the course of an administration of the LMWH, e.g., the anti-Xa to anti-IIa ratio varies no more than about + 1.5, + 1, +0.5, or +0.2, over a period of about 30, 60, 120, 180, 240, 300 minutes.
• an initial ratio of anti-Xa activity to anti-IIa activity is 2
• the ratio measured at a second time e.g., 30, 60, 120, 180, 240, 300 minutes
• the initial administration will preferably be less than 3, and preferably at or around 2.
• the LMWH composition has the following structure:
• the LMWH composition has the following properties: anti-Xa activity of about 100 to 400 IU/mg; anti-Xa activity that is at least 50% neutralizable, e.g., as measured by anti-Xa activity, ACT or aPTT; a polydispersity of less than 1.6; less than 70%, 60%, 50% of the chains present in the composition have a molecular weight greater than 7500 Da; less than 40% of the chains present in the composition have a molecular weight less than 5000 Da; it includes a mixture of ⁇ U and I/G structures at the non-reducing ends of the chains; it has substantially no modified reducing end structures; fewer chains in the composition have PF4 binding sites than enoxaparin, dalteparin, or UFH; at least 60%, 70%, 80%, 90% of the chains of the composition have HNAc at the reducing end; about 15% to 35% of the chains in the composition have a ⁇ U at the non-reducing end; less than 90%, 95%,
• the LMWH composition has the following properties: it has a higher degree of sulfation than enoxaparin or dalteparin; it has more trisulfated disaccharides present in the composition than enoxaparin or dalteparin, e.g., the LMWH composition has about 50 to 65% trisulfated disaccharides, as determined by mole% it has a higher level of ⁇ UH NAC,6S GH NSI3SI6S than enoxaparin, daltaparin and/or UFH, e.g., ⁇ UH N Ac,6sGH N s,3S,6S is present at about 5 to 15 mole%.
• the LMWH composition has the following properties: it has a calcium content less than 3% and/or a sodium content less than 20%; it includes less than 1000 ng/mg of a heparinase enzyme; it has less than 1.0% w/w methanol; it has less than 1.0% w/w ethanol; it has less than 2.0% chloride; it has less than 15% water by weight; it has less than 2000 ppm of free sulfate.
• the LMWH composition has the following properties: it provides increased tissue factor pathway inhibitor (TFPI) release as compared to enoxaparin.
• TFPI tissue factor pathway inhibitor
• the LMWH composition has an intravenous half life of about 30 minutes to 3 hours.
• the invention features, a method of making a LMWH.
• the method includes: subjecting UFH to one, or a step-wise series, of aqueous alcohol (e.g., ethanol) precipitations (at least one with a sodium salt (or a salt other than a calcium salt)), to extract a lower molecular weight fraction from the unfractionated heparin (e.g., the fast moving fraction) to provide a first intermediate, wherein the first intermediate preferably has a average chain length of 10 to 16 disaccharides; digesting the first intermediate using an agent, e.g., an enzyme or chemical, that cleaves glycosidic linkages of unsulfated uronic acids, e.g., an enzyme described herein, e.g., in aqueous buffer, e.g., in aqueous salt buffer, e.g., a sodium acetate buffer, pH of about 5-9, e.g., 7-8, at 25°C to 52°C,e
• the invention features, a LMWH composition made by a method described herein.
• the method includes an intermediate or reaction mixture from any of the methods for making or analyzing a LMWH described herein.
• the invention features, a pharmaceutical composition that includes a LMWH composition described herein.
• the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
• the pharmaceutical composition is in a form suitable for systemic administration.
• the pharmaceutical composition is suitable for subcutaneous, intravenous, intra-arterial, intrasynoval, intramuscular, intraperitoneal, intravitreous, epidural, subdural or intrathecal administration.
• a pharmaceutical composition for systemic administration can be an isotonic solution, e.g., an isotonic solution with or without preservatives. Examples of preservative include, but are not limited to, benzyl alcohol, mannitol and leucine.
• a unit dosage amount of a pharmaceutical composition of the invention can be disposed within a package or a device suitable for administration.
• a composition suitable for subcutaneous delivery can be disposed within a syringe configured for subcutaneous delivery
• a composition suitable for intravenous delivery can be disposed within a syringe configured for intravenous delivery or within another device for intravenous delivery, e.g., an intravenous drip bag or bottle.
• the pharmaceutical composition is in a form suitable for local invasive administration, e.g., coating or within a device suitable for implantation.
• devices suitable for implantation include, but are not limited to, a stent, and an excorporeal circuit.
• the pharmaceutical composition is in a form suitable for subcutaneous implantation, implantation into a tissue or organ (e.g., a coronary artery, carotid artery, renal artery, other peripheral arteries, veins, kidney, heart, cornea, vitreous, and cerebrum), or implantation into a space surrounding a tissue or organ (e.g., kidney capsule, pericardium, thoracic or peritoneal space.
• a tissue or organ e.g., a coronary artery, carotid artery, renal artery, other peripheral arteries, veins, kidney, heart, cornea, vitreous, and cerebrum
• a space surrounding a tissue or organ e.g., kidney capsule, pericardium, thoracic or
• the pharmaceutical composition is in a form suitable for noninvasive administration, e.g., topical, transdermal, pulmonary, nasal, oral, auditory canal, rectal or vaginal administration.
• a unit dosage amount of a pharmaceutical composition of the invention can be disposed within a package or a device suitable for such administration.
• the LMWH composition is lyophilized. In another embodiment, the LMWH composition is a liquid.
• the invention features a container, e.g., an ampoule, syringe or vial, containing the pharmaceutical composition.
• the LMWH composition is present at about 1500IU, 2000IU, 2500 IU, 3000 IU, 3500 IU, 4000 IU, 4500 IU, 5000 IU, 5500 IU, 6000 IU anti-Xa activity per mL pharmaceutically acceptable carrier.
• the pharmaceutical composition has an osmolality of about 200 to 400 mOsm/L, e.g., about 250 to 350 mOsm/L, about 280 to 330 mOsm/L.
• the pharmaceutical composition further comprises sodium chloride and water.
• the invention features, a method of treating a subject including administering a LMWH disclosed herein to the subject.
• the treatment can be therapeutic, e.g., a treatment which lessens, mitigates or ameliorates an existing unwanted condition or symptom thereof, or prophylactic, e.g., a treatment which delays, e.g., prevents, the onset of an unwanted condition or symptom thereof.
• a LMWH composition described herein can be used to treat disorders which are treatable with UFH or with a commercial LMWH, e.g., enoxaparin, daltaparin or tinzaparin.
• the invention includes methods for treating a subject having, or at risk of having, a disorder or condition selected from the group consisting of: a disorder associated with coagulation, e.g., deep vein thrombosis (DVT) or pulmonary embolism, thrombosis or cardiovascular disease, e.g., acute coronary syndrome (ACS), stable or unstable angina, myocardial infarction, e.g., ST-segment elevated myocardial infarction (STEMI) or non-ST-segment elevated myocardial infarction (NSTEMI), vascular conditions or atrial fibrillation; migraine; atherosclerosis; an inflammatory disorder, such as autoimmune disease or atopic disorders, psoriasis, arthritis, sepsis; disseminated intravascular coagulopathy (DIC); an allergy or a respiratory disorder, such as asthma, emphysema, adult respiratory distress syndrome (ARDS), cystic fibrosis, or lung reperfusion injury; stenosis
• the subject can be undergoing, or have undergone, a surgical procedure, e.g., organ transplant, orthopedic surgery, joint replacement, e.g., hip replacement or knee replacement, percutaneous coronary intervention (PCI), stent placement, angioplasty, or coronary artery bypass graft surgery (CABG).
• a surgical procedure e.g., organ transplant, orthopedic surgery, joint replacement, e.g., hip replacement or knee replacement, percutaneous coronary intervention (PCI), stent placement, angioplasty, or coronary artery bypass graft surgery (CABG).
• PCI percutaneous coronary intervention
• stent placement e.g., angioplasty
• CABG coronary artery bypass graft surgery
• CABG coronary artery bypass graft surgery
• the method further includes administering protamine sulfate after administration of the LMWH composition to neutralize some or all of the activity, e.g., anti-Xa activity and/or anti-IIa activity, of the LMWH composition.
• the activity e.g., anti-Xa activity and/or anti-IIa activity
• the method further includes administering protamine sulfate after administration of the LMWH composition to neutralize some or all of the activity, e.g., anti-Xa activity and/or anti-IIa activity, of the LMWH composition.
• about 50%,60%, 70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWH composition is neutralized, e.g., about 50%, 60%, 70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWH composition is neutralized within 5, 10, 15, 20, 25, 30, 40 minutes after protamine administration.
• about 50%, 60%, 70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWH composition is neutralized, e.g., about 50%, 60%, 70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWH composition is neutralized within 5, 10, 15, 20, 25, 30, 40 minutes after protamine administration.
• protamine sulfate is administered at a dose of about 1 mg, 2 mg, 3 mg, 5 mg of the LMWH composition per 100 anti-Xa IU of plasma.
• Neutralization of anti-Xa activity and/or anti-IIa activity can be determined, e.g., by ACT and/or aPTT.
• the invention features, a method of treating (e.g., therapeutically or prophylactically treating) a disorder, e.g., a thrombotic disorder, in a subject.
• the method includes administering a LMWH composition described herein, to thereby treat, preferably prevent, the disorder.
• the disorder is one or more of ACS, myocardial infarction, e.g., NSTEMI OR STEMI, stable angina and unstable angina.
• the thrombotic disorder is arterial thrombosis, e.g., including STEMI.
• the disorder can be, e.g., associated with surgical intervention, e.g., PCI, stent placement or angioplasty.
• the subject can have, or be at risk of having, or be recovering from, a surgical intervention, e.g., cardiology intervention (e.g., angioplasty, PCI, stent placement).
• a surgical intervention e.g., cardiology intervention (e.g., angioplasty, PCI, stent placement).
• the subject is at risk for (e.g., is being considered for) receiving surgical intervention, e.g., CABG.
• the LMWH composition is administered to the subject intravenously, e.g., at a dose of about 0.03 mg/kg to 0.45 mg/kg, e.g., 0.03 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.37 mg/kg, 0.4 mg/kg, 0.44 mg/kg.
• the LMWH composition is administeredintravenously at a dose of about 0.1 to 0.3 mg/kg, e.g., 0.1 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg or 0.30 mg/kg.
• the LMWH composition is administered to the subject subcutaneously, e.g., at a dose of about 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.44 mg/kg, 0.47 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg.
• the LMWH composition is administered subcutaneously at a dose of about 0.15 to 1.0 mg/kg, 0.20 to 0.9 mg/kg, 0.25 to 0.9 mg/kg, 0.30 to 0.50 mg/kg, e.g., 0.30mg/kg, 0.35mg/kg, 0.40 mg/kg, 0.42 mg/kg, 0.44 mg/kg, 0.47 mg/kg or 0.50 mg/kg.
• the method further includes monitoring the activity of the LMWH composition in the subject using a coagulation assay, e.g., using ACT and/or aPTT.
• anti-Xa activity and/or anti-IIa activity is monitored, e.g., with ACT and/or aPTT, prior to, during, or after surgical intervention, e.g., angioplasty, PCI, stent placement.
• anti-Xa activity and/or anti-IIa activity is monitored, e.g., with ACT and/or aPTT, prior to, during, and/or after administration of the LMWH composition.
• anti-Xa activity and/or anti-IIa activity is monitored by ACT, and the dose of LMWH is administered to achieve an ACT of about 200 to 350 seconds.
• the method further includes administering protamine sulfate after administration of the LMWH composition to neutralize some or all of the activity, e.g., anti-Xa activity and/or anti-IIa activity, of the LMWH composition.
• the activity e.g., anti-Xa activity and/or anti-IIa activity
• at least about 50%,60%, 70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWH composition is neutralized, e.g., at least about 50%,60%, 70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWH composition is neutralized within 5, 10, 15, 20, 25, 30, 40 minutes after protamine administration.
• At least about 50%, 60%, 70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWH composition is neutralized, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95% or all of the anti-IIa activity of the LMWH composition is neutralized within 5, 10, 15, 20, 25, 30, 40 minutes after protamine administration.
• protamine sulfate is administered at a dose of about 1-5 mg, e.g., 1 mg, 2 mg, 3 mg, 5 mg of the LMWH composition per 100 anti-Xa IU of plasma.
• Neutralization of anti-Xa activity and/or anti- Ha activity can be determined, e.g., by ACT and/or aPTT.
• anti-Xa activity and/or anti-IIa activity can be determined, e.g., by ACT and/or aPTT, prior to, during and/or after administration of protamine sulfate.
• anti-Xa activity and/or anti-IIa activity is neutralized prior to, during or after surgical intervention.
• anti-Xa activity and/or anti-IIa activity can be neutralized during or after a surgical intervention such as angioplasty or PCI.
• the LMWH composition is neutralized prior to surgical intervention such as CABG.
• the method further includes monitoring the patient for a negative reaction, e.g., epidural or spinal hematoma, hemorrhage or bleeding.
• a negative reaction e.g., epidural or spinal hematoma, hemorrhage or bleeding.
• the LMWH composition is administered intravenously or subcutaneously.
• the LMWH composition is administered in combination with another therapeutic agent, e.g., an anticoagulant or antithrombotic agent, e.g., bivalirudin Angiomax), ASA, a GPIIbIIIa inhibitor (e.g., eptif ⁇ batide or abciximab), an ADP inhibitor (e.g., Plavix), rPA, TNKase, aspirin, a P2Y12 inhibitor, a platelet inhibitor, warfarin, and combinations thereof.
• an anticoagulant or antithrombotic agent e.g., bivalirudin Angiomax
• ASA e.g., bivalirudin Angiomax
• GPIIbIIIa inhibitor e.g., eptif ⁇ batide or abciximab
• an ADP inhibitor e.g., Plavix
• rPA TNKase
• aspirin e.g., aspirin, a P2Y12 inhibitor, a platelet inhibitor,
• the invention features, a method of treating (e.g., therapeutic or prophylactic treatment) a disorder, e.g., a thrombotic or cardiovascular disorder, in a patient.
• a disorder e.g., a thrombotic or cardiovascular disorder
• the method includes: optionally, administering a reversible and monitorable LMWH composition described herein to the patient; classifying the patient (to whom the LMWH composition has been or will be administered) as not in need of surgical intervention (e.g., classifying the patient as not in need of surgical intervention prior to release from the hospital) or as a candidate for surgical intervention prior to release from the hospital; optionally, if the patient is classified as a candidate for surgical intervention then performing one or both of monitoring (e.g., as described herein, e.g., with a coagulation assay, e.g., using ACT and/or aPTT) the reversible and monitorable LMWH composition and neutralizing (e.g., as described herein, e.g., by administering protamine sulfate) the reversible and monitorable LMWH composition.
• monitoring e.g., as described herein, e.g., with a coagulation assay, e.g., using
• the LMWH composition is administered to the subject intravenously, e.g., at a dose of about 0.03 mg/kg to 0.45 mg/kg, e.g., 0.03 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.37 mg/kg, 0.4 mg/kg, 0.44 mg/kg.
• the LMWH composition is administeredintravenously at a dose of about 0.1 to 0.3 mg/kg, e.g., 0.1 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg or 0.30 mg/kg.
• the LMWH composition is administered to the subject subcutaneously, e.g., at a dose of about 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.44 mg/kg, 0.47 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg.
• the LMWH composition is administered subcutaneously at a dose of about 0.15 to 1.0 mg/kg, 0.20 to 0.8 mg/kg, 0.25 to 0.90 mg/kg, 0.30 to 0.50 mg/kg, e.g., 0.30mg/kg, 0.35mg/kg, 0.40 mg/kg, 0.42 mg/kg, 0.44 mg/kg, 0.47 mg/kg or 0.50 mg/kg.
• the patient is classified as a candidate for surgical intervention, e.g., PCI, stent placement or angioplasty, and the effect of the reversible and monitorable LMWH composition is monitored.
• the surgical intervention is performed and the reversible and monitorable LMWH composition is monitored at one or more, or all, of before, during and after the surgery.
• the patient is monitored for an ACT of about 200 to 350 before and/or during a surgical intervention such as PCI.
• the patient is classified as a candidate for surgical intervention, e.g., CABG and the effect of the reversible and monitorable LMWH composition is one or both of monitored and neutralized.
• the surgical intervention is performed and the reversible and monitorable LMWH composition monitored one or more, or all of before, during and after the surgery.
• the patient is monitored for an ACT of about 400 to 600, e.g., 400 to 500 prior to surgical intervention such as CABG.
• the subject is being treated for a thrombotic disorder.
• the disorder is one or more of ACS, myocardial infarction, e.g., NSTEMI OR STEMI, stable angina and unstable angina.
• the thrombotic disorder is arterial thrombosis, e.g., including ST elevation (STEMI).
• the invention features, a method of monitoring a subject treated with a monitorable LMWH composition described herein.
• the method includes, optionally, administering a monitorable LMWH composition described herein to the subject; and evaluating aPTT and/or ACT in the subject (who has been administered the monitorable LMWH composition).
• a baseline aPTT and/or ACT is determined prior to treating the subject with the LMWH.
• the method includes comparing aPTT and/or ACT of a subject that has received the LMWH to the baseline aPTT and/or ACT.
• the subject is monitored at one or more, or all, of the following stages: prior to, during and after receiving a LMWH composition.
• the subject is monitored prior to, during and/or after surgical intervention, e.g., PCI, stent placement or angioplasty.
• the LMWH composition is monitored for an ACT of about 200 to 350 prior to and/or during a surgical intervention such as PCI.
• the LMWH composition is monitored for an ACT of about 400 to 600, e.g., about 400 to 500, prior to CABG.
• the invention features, a method of treating a subject who has been administered a reversible LMWH composition described herein.
• the method includes: optionally, administering a reversible LMWH composition described herein to the subject; and neutralizing (e.g., as described herein, e.g., by administering protamine sulfate) the reversible LMWH composition.
• the subject is monitored at one or more, or all, of the following stages: prior to, during and after administration of protamine sulfate.
• the invention features, a method of advising on, or providing instructions (e.g., written, oral, or computer generated instructions) for, the use of a LMWH having high anti-IIa activity, e.g., a LMWH composition described herein.
• the method includes providing instruction regarding use, e.g., with: patients having abnormal renal function or diabetes or clot bound thrombin; patients who are candidates for PCI, stent placement, CABG, angioplasty, etc.; interventional cardiology patients; patients in need of neutralization of previously administered LMWH, e.g., neutralizing with protamine sulfate; patients at risk of epidural or spinal hematoma, hemorrhage and/or bleeding.
• the instruction pertains to administration of the LMWH composition for ACS, myocardial infarction, e.g., NSTEMI OR STEMI, stable angina and unstable angina, e.g., administration in a sub population of patients such as patients having abnormal renal function, or elderly patients (e.g., patients over 60 years of age).
• the instruction pertains to administration of the LMWH composition for thrombotic disorders, e.g., thrombotic disorders associated with surgical intervention, e.g., PCI, stent replacement or angioplasty.
• the invention features, a method of advising on the use of a LMWH composition described herein, that includes providing instruction regarding monitoring anti-Xa activity and/or anti-IIa activity using ACT and/or aPTT.
• the invention features, a method of manufacturing a LMWH composition, e.g., a LMWH composition described herein.
• the method includes one or more of the following steps:
• glycosaminoglycan containing sample, e.g., UFH
• a glycosaminoglycan (GAG) containing sample e.g., UFH
• a first a precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), a polar non-organic solvent (e.g., water), and a salt (e.g., a sodium salt, e.g., sodium acetate, or calcium salt, e.g., calcium acetate), to yield a first supernatant;
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• a salt e.g., a sodium salt, e.g., sodium acetate, or calcium salt, e.g., calcium acetate
• a second precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and a polar non-organic solvent (e.g., water), to yield a precipitate (this can be used to provide a fast moving fraction as discussed elsewhere herein);
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• solublizing the precipitate preferably in water, and cleaving the solubilized precipitate with an agent that cleaves glycosidic linkages of unsulfated uronic acids, e.g., adjacent to an N-acetyl glucosamine residue.
• an agent that cleaves glycosidic linkages of unsulfated uronic acids e.g., adjacent to an N-acetyl glucosamine residue.
• An example is a heparinase III enzyme described herein, preferably MOl 1, preferably in the presence of sodium acetate, and preferably to completion, e.g., as indicated by a UV plateau, to provide a cleaved preparation;
• cleaved preparation e.g., with a salt, e.g., a sodium salt, preferably sodium chloride, and a polar organic solvent, e.g., an alcohol, e.g., methanol, to form solids, having saccharides with e.g., an average chain length of 8-14, e.g., 8-12 dissacharides;
• a salt e.g., a sodium salt, preferably sodium chloride
• a polar organic solvent e.g., an alcohol, e.g., methanol
• a purification step e.g., a chromatographic purification step, e.g., size selection step, e.g., exclusion chromatography, ion exchange chromatography, or filtration, to provide a preparation with a higher average molecular weight than in step (4).
• the higher average molecular weight preparation has an average chain length of 9 to 16 dissacharides or an average molecular weight of 5000 to 9000 Da.
• the invention features, methods of making a LMWH composition having an average chain length of about 9 to 16 disaccharides.
• the method includes: providing a precursor LMWH composition (e.g., a intermediate composition from a method described herein) having an average chain length of less than 9 to 16 disaccharides, preferably about 8 to 14 disaccharides, e.g., 8 to 12 disaccharides; and processing the precursor LMWH composition to obtain a LMWH having an average chain length of about 9 to 16 disaccharides.
• the processing includes size-based selection, e.g., a size-dependent separation, e.g., by one or more of size exclusion chromatography, ion exchange chromatography or filtration.
• the precursor LMWH composition is a preparation having an average chain length of about 8 to 14 disaccharides, e.g., 8 to 12 disaccharides. In a preferred embodiment it was obtained by a method including salt precipitation and enzymatic digestion of a higher molecular weight preparation, e.g., UFH.
• the salt is a salt of a monovalent or divalent cation. Examples of monovalent and divalent cations that can be used include, e.g., sodium, potassium, rubidium, cesium, barium, calcium, magnesium, strontium, and combinations thereof.
• the salt of monovalent or divalent cation is an acetate of a monovalent or divalent cation.
• the enzyme (or enzymes) used for digestion cleaves at one or more glycosidic linkages of unsulfated uronic acids, e.g., adjacent to an N-acetyl glucosamine residue.
• examples of enzymes that can be used include, e.g., heparinase III, mutants of heparinase III, e.g., a heparinase III mutant described in U.S.
• Patent Number 5,896,789 e.g., a mutant of heparinase III having one or more histidine residue selected from the group consisting of His 36, HislO5, Hisl 10, Hisl39, Hisl52, His225, His234, His241, His424, His469, and His539 has been substituted with an alanine
• heparin sulfate glycosaminoglycan lyase III from Bacteroides thetaiotaomicron.
• the enzyme used for digestion is a mutated heparinase III having an alanine at residue 225 of the amino acid sequence substituted with an alanine.
• the precursor composition can be obtained by:
• glycosaminoglycan containing sample, e.g., UFH
• a glycosaminoglycan (GAG) containing sample e.g., UFH
• a first a precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), a polar non-organic solvent (e.g., water), and a salt (e.g., a sodium salt, e.g., sodium acetate, or a calcium salt, e.g., calcium acetate), to yield a first supernatant;
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• a salt e.g., a sodium salt, e.g., sodium acetate, or a calcium salt, e.g., calcium acetate
• a second precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and a polar non-organic solvent (e.g., water), to yield a precipitate (this can be used to provide a fast moving fraction as discussed elsewhere herein); (3) solublizing the precipitate and cleaving the solubilized precipitate with a heparinase III enzyme, preferably MOl 1, preferably in the presence of sodium acetate, and preferably to completion as, e.g., indicated by UV absorption of greater than 9.8, to provide a cleaved preparation.
• a heparinase III enzyme preferably MOl 1, preferably in the presence of sodium acetate, and preferably to completion as, e.g., indicated by UV absorption of greater than 9.8, to provide a cleaved preparation.
• the invention features a method of making a LMWH composition, e.g., a LMWH composition described herein.
• the method includes:
• glycosaminoglycan containing sample, e.g., UFH
• a first a precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), a polar non-organic solvent (e.g., water), and a sodium salt (e.g., sodium acetate), to yield a first supernatant
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• sodium salt e.g., sodium acetate
• a second precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and a polar non-organic solvent (e.g., water), to yield a precipitate (this can be used to provide a fast moving fraction as discussed elsewhere herein);
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• the enzyme (or enzymes) used for digestion cleaves at one or more glycosidic linkages of unsulfated uronic acids, e.g., adjacent to an N-acetyl glucosamine residue.
• examples of enzymes that can be used include, e.g., heparinase III, mutants of heparinase III, e.g., a heparinase III mutant described in U.S.
• Patent Number 5,896,789 e.g., a mutant of heparinase III having one or more histidine residue selected from the group consisting of His 36, HislO5, Hisl 10, Hisl39, Hisl52, His225, His234, His241, His424, His469, and His539 has been substituted with an alanine
• heparin sulfate glycosaminoglycan lyase III from Bacteroides thetaiotaomicron.
• the enzyme used for digestion is a mutated heparinase III having an alanine at residue 225 of the amino acid sequence substituted with an alanine.
• the digested fraction is the final product.
• the method can include one or more additional processing steps to obtain a final product.
• the method includes processing the digested fraction to obtain a LMWH composition having an average chain length of 9 to 16 disaccharides.
• size exclusion chromatography, ion exchange chromatography and/or filtration can be used to obtain a LMWH composition having an average chain length of 9 to 16 disaccharides.
• the invention features, methods of evaluating or processing a GAG such as UFH to determine suitability of the GAG for processing into a LMWH composition, e.g., a LMWH composition described herein.
• the method includes determining the quantity of N-acetyl present in a GAG preparation, comparing the quantity to a preselected criterion and making a decision about the GAG preparation based upon the whether the preselected criterion is met.
• a decision or step is taken, e.g., the GAG preparation is classified, accepted or discarded, processed into a drug substance or drug product, or a record made or altered to reflect the determination, depending upon whether the preselected criterion is met.
• a decision can be made about altering one or more steps in manufacturing of a LMWH composition.
• the preselected criterion is N-acetyl present in the GAG preparation at an amount of about 11% or higher, e.g., as determined by mole %, relative to total glucosamine content.
• a GAG preparation having N-acetyl content within this range is indicative of a GAG preparation suitable for processing into a LMWH composition, e.g., a LMWH composition described herein.
• this preselected criterion is met, the GAG preparation is accepted and processed into intermediates, drug substance or drug product.
• the amount of N-acetyl present in a GAG preparation can be determined using, e.g., nuclear magnetic resonance (NMR).
• NMR nuclear magnetic resonance
• methods disclosed herein are useful from a process standpoint, e.g., to monitor or ensure batch-to-batch consistency or quality, or to evaluate a sample with regard to a preselected criterion.
• the invention features, a method of evaluating or processing an intermediate LMWH preparation, e.g., produced by a method described herein, to determine suitability of the intermediate preparation for processing into a LMWH composition.
• the intermediate LMWH preparation is a fast moving fraction obtained, e.g., by salt precipitation with sodium or sodium acetate of a glycosaminoglycan (GAG) containing sample in a solvent as described herein.
• GAG glycosaminoglycan
• the method includes comparing the quantity of one or more of structural moieties, e.g., one or more of sulfated iduronic acid, N-sulfated hexosamine linked to uronic acid, epoxide and 6-0 sulfated hexosamine, in the intermediate LMWH preparation to the quantity of the same structural moiety in unfractionated heparin starting material, and making a decision about the intermediate LMWH preparation based upon whether a preselected criterion between the starting material and intermediate LMWH preparation is met.
• structural moieties e.g., one or more of sulfated iduronic acid, N-sulfated hexosamine linked to uronic acid, epoxide and 6-0 sulfated hexosamine
• a decision or step is taken, e.g., the intermediate LMWH preparation is classified, accepted or discarded, processed into a drug substance or drug product, or a record made or altered to reflect the determination, depending upon whether the a preselected relationship is met.
• a decision can be made about altering one or more steps in manufacturing of a LMWH composition.
• the preselected criterion is a decrease in sulfated iduronic acid in the intermediate preparation as compared to the starting material.
• An intermediate preparation having a decreased sulfated iduronic acid content is indicative of an intermediate preparation suitable for further processing into a LMWH composition, e.g., a LMWH composition described herein.
• this preselected criterion is met, the intermediate preparation is accepted and processed into further intermediates, drug substance or drug product.
• the preselected criterion is an increase in N-sulfated hexosamine linked to uronic acid (e.g., iduronic and/or glucuronic acid) in the intermediate preparation as compared to the starting material.
• An intermediate preparation having an increased N-sulfated hexosamine linked to uronic acid is indicative of an intermediate preparation suitable for further processing into a LMWH composition, e.g., a LMWH composition described herein.
• this preselected criterion is met, the intermediate preparation is accepted and processed into further intermediates, drug substance or drug product.
• the preselected criterion is a decrease in epoxide in the intermediate preparation as compared to the starting material.
• An intermediate preparation having a decreased epoxide content is indicative of an intermediate preparation suitable for further processing into a LMWH composition, e.g., a LMWH composition described herein.
• this preselected criterion is met, the intermediate preparation is accepted and processed into further intermediates, drug substance or drug product.
• the preselected criterion is an increase in 6-0 sulfated hexosamine in the intermediate preparation as compared to the starting material.
• An intermediate preparation having increased 6-0 sulfated hexosamine is indicative of an intermediate preparation suitable for further processing into a LMWH composition, e.g., a LMWH composition described herein.
• this preselected criterion is met, the intermediate preparation is accepted and processed into further intermediates, drug substance or drug product.
• the amount of a structural moiety in the starting material and/or intermediate preparation is determined using one or more of nuclear magnetic resonance (NMR), capillary electrophoresis (CE) and high performance liquid chromatography (HPLC).
• NMR nuclear magnetic resonance
• CE capillary electrophoresis
• HPLC high performance liquid chromatography
• methods disclosed herein are useful from a process standpoint, e.g., to monitor or ensure batch-to-batch consistency or quality, or to evaluate a sample with regard to a preselected criterion.
• the invention provides a method of evaluating a UFH preparation as a starting material to make a LMWH composition described herein.
• the method includes providing an evaluation of the UFH preparation for a parameter related to suitability of the UFH sample for use in the making of a LMWH described herein; and optionally, providing a determination of whether a value (e.g., a value correlated to presence, amount, distribution, or absence) determined for the parameter meets a preselected criterion, e.g., is present, or is present within a preselected range, thereby evaluating the UFH sample.
• a value e.g., a value correlated to presence, amount, distribution, or absence
• the criterion is satisfied and the UFH sample is selected and processed into the LMWH.
• the parameter is the presence or amount of a structure listed in Table 2, preferably one related to the efficacy of a step in the method of making the LMWH, e.g., a structure which promotes or is positively correlated with cleavage by a heparinase, e.g., H NAc (internal).
• a structure which promotes or is positively correlated with cleavage by a heparinase e.g., H NAc (internal).
• the method includes determining if the amount of HNAC (internal) in the UFH sample has a predetermined relationship with a reference, e.g., it is equal to or greater than a preselected reference value.
• a value for the parameter in an intermediate used in making the LMWH is also determined and optionally, that value must also meet a predetermined criterion to select the UFH for use in making the LMWH.
• the invention provides a method of evaluating a UFH preparation, as a starting material to make a LMWH composition described herein.
• the method includes optionally, performing an operation, e.g., a precipitation, on the UFH sample to provide an intermediate (preferably the steps used to produce this intermediate and the intermediate are the same as the steps and an intermediate of the method used to make the LMWH); providing an evaluation of the intermediate preparation for a parameter related to suitability of the UFH sample for use in the making of a LMWH described herein; and optionally, providing a determination of whether a value (e.g., a value correlated to presence, amount, distribution, or absence) determined for the parameter meets a preselected criterion, e.g., is present, or is present within a preselected range, thereby evaluating the UFH preparation.
• an operation e.g., a precipitation
• the criterion is satisfied and the UFH sample is selected and processed into the LMWH.
• the parameter is the presence or amount of a structure listed in Table 2, preferably one related to the efficacy of a step in the method of making the LMWH, e.g., a structure which promotes or is positively correlated with cleavage by a heparinase, e.g., H NAc (internal).
• a structure which promotes or is positively correlated with cleavage by a heparinase e.g., H NAc (internal).
• the method includes determining if the amount of HNAC (internal) in the intermediate sample has a predetermined relationship with a reference, e.g., it is equal to or greater than a preselected reference.
• a value for the parameter in the UFH is also determined and optionally, that value must also meet a predetermined criterion to select the UFH for use in making the LMWH.
• a decision or step is taken, e.g., the sample is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, labeled, packaged, released into commerce, or sold or offered for sale, or a record made or altered to reflect the determination, depending on whether the preselected criterion is met.
• the batch from which the sample is taken can be processed, e.g., as just described.
• a preferred embodiment includes analyzing the sample with NMR.
• either method can include providing a comparison of the value determined for a parameter with a reference value or values, to thereby evaluate the sample.
• the comparison includes determining if the test value has a preselected relationship with the reference value, e.g., determining if it meets the reference value.
• the value need not be a numerical value but, e.g., can be merely an indication of whether the subject entity is present.
• a preferred embodiment of either method can include determining if a test value is equal to or greater than a reference value, if it is less than or equal to a reference value, or if it falls within a range (either inclusive or exclusive of one or both endpoints).
• test value or an indication of whether the preselected criterion is met, can be memorialized, e.g., in a computer readable record.
• intermediate is prepared by one or more or all of the following steps:
• a precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), a polar non-organic solvent (e.g., water), and a salt (e.g., a sodium salt, e.g., sodium acetate), to yield a first supernatant;
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• a salt e.g., a sodium salt, e.g., sodium acetate
• a second precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), and a polar non-organic solvent (e.g., water), to yield a precipitate (this can be used to provide a fast moving fraction as discussed elsewhere herein);
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• the preferred intermediate is that produced in step (2) or (3a), though the method can use others.
• the invention features, a method of evaluating a LMWH preparation described herein.
• the method includes: providing a LMWH preparation described herein; determining if a structure, activity or function described herein is present in or possessed by the preparation, thereby evaluating a LMWH preparation described herein.
• the determining includes determining if the structure, activity or function is present at a preselected level or in a preselected range, e.g., a level or range disclosed herein.
• the invention provides a method of evaluating or processing a LMWH composition described herein.
• the method includes: providing an evaluation of a parameter related to a peak listed in Table 1OA.
• Such parameters can include, or be a function of, the presence, relative distribution, or amount of a peak, and, optionally, providing a determination of whether a value (e.g., a value correlated to presence, amount, distribution, or absence) determined for the parameter meets a preselected criterion, e.g., is present, or is present within a preselected range, thereby evaluating or processing the mixture.
• the method includes analyzing, e.g., separating, a digest of the sample by digestion with heparinase I, heparinase II, heparinase III by electrophoresis, e.g., capillary electrophoresis.
• the method includes evaluating a sample to determine if one or more of the peaks listed in Table 1OA is present.
• the method includes providing a comparison of the value determined for a parameter with a reference value or values, to thereby evaluate the sample.
• the comparison includes determining if the test value has a preselected relationship with the reference value, e.g., determining if it meets the reference value.
• the value need not be a numerical value but, e.g., can be merely an indication of whether the subject entity is present.
• the method includes determining if a test value is equal to or greater than a reference value, if it is less than or equal to a reference value, or if it falls within a range (either inclusive or exclusive of one or both endpoints).
• the amount of a peak listed in Table 1OA can be determined and, optionally shown to fall within a preselected range, e.g., a range which corresponds to a range from Table 1OA.
• the amount of each peak is about that found in Table 1OA, the amount of each peak is within a range provided in Table 1OA; the amount of peaks 10 and 11 are with a range provided in Table 1OA.
• test value or an indication of whether the preselected criterion is met, can be memorialized, e.g., in a computer readable record.
• a decision or step is taken, e.g., the sample is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, labeled, packaged, released into commerce, or sold or offered for sale, or a record made or altered to reflect the determination, depending on whether the preselected criterion is met. E.g., based on the result of the determination or whether one or more subject entities is present, or upon comparison to a reference standard, the batch from which the sample is taken can be processed, e.g., as just described.
• the structures in Table 1OA can be determined using CE, and when necessary, by other analytical methods.
• the invention features, a method of evaluating or processing a LMWH preparation described herein.
• the method includes: providing a LMWH preparation which has been digested with heparinase I, heparinase II, heparinase III and separated by a separation technique such as CE; determining if one or more of the peaks listed in Table 1OA is present.
• the method includes determining if a peak listed in Table 1OA falls within a preselected range from Table 1OA. In a preferred embodiment: the amount of each peak is about that found in Table 1OA, the amount of each peak is within a range provided in Table 1OA; the amount of peaks 10 and 11 are with a range provided in Table 1OA.
• the invention provides a method of evaluating or processing a LMWH composition described herein.
• the method includes providing an evaluation of a parameter related to the structure or structures of Table 1 IA.
• a parameter related to the structure or structures of Table 1 IA can include, or be a function of, the presence, relative distribution, or amount of a structure, and, optionally, providing a determination of whether a value (e.g., a value correlated to presence, amount, distribution, or absence) determined for the parameter meets a preselected criterion, e.g., is present, or is present within a preselected range, thereby evaluating or processing the mixture.
• a value e.g., a value correlated to presence, amount, distribution, or absence
• the method includes analyzing the composition using 2D-NMR.
• the method includes evaluating a sample to determine if one or more of the structures provided in Table 1 IA is present.
• the method includes providing a comparison of the value determined for a parameter with a reference value or values, to thereby evaluate the sample.
• the comparison includes determining if the test value has a preselected relationship with the reference value, e.g., determining if it meets the reference value.
• the value need not be a numerical value but, e.g., can be merely an indication of whether the structure is present.
• the method includes determining if a test value is equal to or greater than a reference value, if it is less than or equal to a reference value, or if it falls within a range (either inclusive or exclusive of one or both endpoints).
• the amount of a structure provided in Table 1 IA can be determined and, optionally shown to fall within a preselected range, e.g., a range which corresponds to a range from Table 1 IA.
• the amount of each structure is about that found in Table 1 IA, the amount of each structure is within a range provided in Table HA.
• test value or an indication of whether the preselected criterion is met, can be memorialized, e.g., in a computer readable record.
• a decision or step is taken, e.g., the sample is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, labeled, packaged, released into commerce, or sold or offered for sale, or a record made or altered to reflect the determination, depending on whether the preselected criterion is met. E.g., based on the result of the determination or whether one or more structures is present, or upon comparison to a reference standard, the batch from which the sample is taken can be processed, e.g., as just described.
• Table 1 IA can be determined using 2D NMR, and when necessary, by other analytical methods.
• the invention features, a method of evaluating or processing a LMWH preparation described herein.
• the method includes providing a LMWH preparation which has been analyzed using 2D NMR; determining if one or more of the structure listed in Table 1 IA is present.
• the method includes determining if a structure listed in Table 1 IA falls within a preselected range, e.g., a range which corresponds to a range 1 from Table 1 IA. In a preferred embodiment: the amount of each structure is about that found in Table 1 IA, the amount of each structure is within a range provided in Table HA.
• Some methods described herein include making a determination of whether a subject entity is present at a preselected level or within a preselected range and that level or range is expressed in specific units of measurement, e.g., mole %, e.g., present in a range of X-Y mole %.
• mole % e.g., present in a range of X-Y mole %.
• the sample has an actual level of subject entity, which can be expressed as X-Y when described in units of mole %.
• That actual level can also be expressed in other units, e.g., weight %. That actual level is the same regardless of the units in which it is expressed.
• the specification of mole % in the method is merely to indicate the actual prevalence of the subject entity.
• the level of subject entity can be measured in terms of other units and the reference value can be expressed in terms of other units, as long as the reference value as expressed in terms of alternative units corresponds to the same amount of subject entity as the reference value expressed in mole %, e.g., X-Ymole % in this example.
• a method which requires showing the subject entity is present at X-Y mole % can be performed by showing that the subject entity is present in a range expressed in an alternative unit of measure, e.g., weight %, chain number, or %AUC, wherein the range, as described in the alternative unit of measure, corresponds to the same amount of subject entity which would give the mole % referred to, in this example X-Y mole %.
• an alternative unit of measure e.g., weight %, chain number, or %AUC
• Figure 1 is a flow chart depicting the four steps of manufacturing process of M118-REH.
• Figure 2A is a graph depicting capillary electrophoresis profile of enoxaparin digested with heparinase I, heparinase II and heparinase III.
• Figure 2B is a graph depicting capillary electrophoresis profile of Ml 18-REH digested with heparinase I, heparinase II and heparinase III.
• Figure 3 is a graph depicting a comparison of the UV and fluorescence profiles generated by post-column labeling for a digest of Ml 18-REH analyzed by Ion-pairing RP HPLC.
• the top trace reflects UV 232nm detection, and the bottom trace reflects fluorescence detection at 410 nm.
• the species labeled by arrows show up mainly in the fluorescence profile and are not observed in the UV profile; they represent non-reducing end saccharides of Ml 18-REH chains that arise from the starting UFH.
• Figure 4 is graph depicting a two-dimensional NMR HSQC analysis of Ml 18- REH.
• Figure 5 is a diagram depicting formation of reducing and non-reducing ends.
• Figure 6 is a manufacturing process flow diagram of Ml 18-REH Injectable.
• Figure 7 is a graph depicting in vitro neutralization of LMWHs by protamine sulfate.
• Ml 18 (shorthand in this figure for Ml 18-REH) is represented by a lighter dot; enoxaparin sodium is represented by a darker dot.
• Graph was plotted with percentage of remaining anti-Xa activity against the ratio of protamine to LMWHs activity.
• Ml 18 which is shorthand for Ml 18-REH in this figure
• Figure 9 is a graph depicting the pharmacodynamics of Ml 18-REH by measuring ACT and aPTT after intravenous injection in the NHP model.
• Figure 10 is a graph depicting comparison of TTO of Ml 18-REH 0.5 (third column) and 1 mg/kg (sixth column) with enoxaparin sodium 2, 3 and 4 mg/kg (second, fourth, and fifth, respectively) intravenously injected in ferric chloride induced thrombosis model. All treatment groups have significant longer TTO compared with Control (first column). There is a statistical significant difference between Ml 18-REH (1 mg/kg) and enoxaparin sodium 3 mg/kg (p ⁇ 0.01).
• Figure 11 is a graph depicting neutralization of anti-Xa activity of heparins in Sprague-Dawley rat model (Ml 18-REH, enoxaparin sodium and UFH ) by protamine sulfate. Graph was plotted with percent of remaining anti-Xa activity vs. time at ratios of 0.5 or 1 mg : 100 anti-Xa IU of protamine to heparin ( or 0.5 or 1 mg: 1 mg in case of UFH ).
• FIG. 13 Anti-Factor Xa activity (top) and anti-Factor Ha activity (bottom) vs. time in a canine model of deep arterial thrombosis (Lucchesi's model).
• the vehicle control group shown in the graphs subsequently received Ml 18-REH at 150 IU/kg. Error bars are ⁇ SE. UFH, unfractionated heparin.
• FIG. 14 Correlation of anti-Factor Xa and Ha activities in a canine model of deep arterial thrombosis (Lucchesi's model). Individual points represent data from a single animal. All animals in all treatment groups are shown. Correlation coefficients (r 2 ) were 0.890 and 0.465 in the Ml 18-REH and unfractionated heparin (UFH) groups, respectively.
• FIG. 16 Coagulation activity vs. time as assessed by ACT ⁇ top), aPTT (middle), and PT (bottom) assays in a canine model of deep arterial thrombosis.
• the vehicle control group shown in the graphs subsequently received Ml 18-REH at 150 IU/kg. Error bars are ⁇ SE.
• UFH unfractionated heparin.
• Figure 17. Percentage of animals with occluded femoral arteries in a canine model of deep arterial thrombosis. Animals were monitored by Doppler flow for up to 180 minutes post current initiation. Occlusion was defined as blood flow through an injured artery that was ⁇ 2% of baseline flow.
• UFH unfractionated heparin.
• LMWH preparations are preferred over UFH preparations because LMWHs have more predictable pharmacokinetics and can be administered subcutaneously.
• currently available LMWH preparations lack many of the desirable properties of UFH such as substantial anti-IIa activity, reversibility (or neutralizability) with protamine sulfate and monitorability.
• LMWHs are not an optimal or practical treatment choice.
• the invention features LMWH preparations designed to have properties that are clinically advantageous, e.g., over other commercially available LMWH preparations and UFH preparations.
• Such properties include, e.g., one or more of: reversibility with proteomine sulfate; predictable pharmacokinetics, anti-IIa activity; substantially constant anti-Xa activity to anti-IIa activity ratio; monitorable activity levels by standard tests such as, e.g., ACT or aPTT; subcutaneous bioavailability; and reduced occurrence of HIT.
• LMWH preparations are disclosed herein that include a significant number of chains of sufficient length (which can be described, e.g., in terms of average chain length of the preparation and/or weight average molecular weight of the preparation) to provide anti-IIa activity, e.g., anti-IIa activity of about 50 to 300 IU/mg, about 70 to 280 IU/mg, about 90 to 250 IU/mg, about 100 to 140 IU/mg, about 100 to 140 IU/mg, about 150 to about 200 IU/mg, about 130 to 190 IU/mg, about 155 to 195 IU/mg.
• Anti-IIa activity is calculated in International Units of anti- Ha activity per milligram using the statistical methods for parallel line assays. The anti-IIa activity levels described herein are measured using the following principle.
• Anti-factor Ha activity is determined by the sample potentiating effect on antithrombin (ATIII) in the inhibition of thrombin. Thrombin excess can be indirectly spectrophotometrically measured.
• the anti-factor Ha activity can be measured, e.g., on a Diagnostica Stago analyzer or on an ACL FuturaTM Coagulation system, with reagents from Chromogenix (S-2238 substrate, Thrombin (53nkat/vial), and Antithrombin), or on any equivalent system. Analyzer response is calibrated using the 2nd International Standard for Low Molecular Weight Heparin.
• a determination of whether a LMWH preparation includes chains of sufficient chain length can be made, for example, by determining the average chain length of the chains in the LMWH preparation and/or by determining the weight average molecular weight of chains within the LMWH preparation.
• average chain length is determined, an average chain length of about 5 to 20, e.g., 7 to 18, preferably about 9 to 16 or 8 to 14 disaccharide repeats, indicates that a significant number of chains in the LMWH preparation are of sufficient chain length.
• Average chain length refers to the average chain length of uronic acid/hexosamine disaccharide repeats that occur within a chain. The presence of non- uronic acid and/or non-hexosamine building blocks (e.g., attached PEG moieties) are not included in determining the average chain length. Average chain length is determined by dividing the number average molecular weight (Mn) by the number average molecular weight for a disaccharide (500 Da). Methods of determining number average molecular weight are described below using SEC MALS.
• LMWH preparations include the following:
• R is H or SO 3 X
• Rl is SO 3 X or COCH 3 and X is a monovalent or divalent cation (e.g., Na or Ca); and average n is about 9 to 16 or 8 to 15;
• R is H or SO 3 X
• Rl is SO 3 X or COCH 3 , X is a monovalent or divalent cation (e.g., Na or Ca); and average n is about 9 to 16 or 8 to 15;
• X is a monovalent or divalent cation (e.g., Na or Ca); R is H or SO 3 X; Rl is SO 3 X or COCH 3 ; and average n is about 8 to 12 or 7 to 11; and
• X is a monovalent or divalent cation (e.g., Na or Ca); R is H or SO 3 X; Rl is SO 3 X or COCH 3 ; average n is 8 to 12 or 7 to 11, and
• a weight average molecular weight of a preparation When weight average molecular weight of a preparation is determined, a weight average molecular weight of about 5000 to 9000 Da, about 5000 to 8300 Da, preferably about 5500 to 8000 Da, about 5700 to 7900, or about 5800 to 6800 Da, indicates that a significant number of chains in the LMWH preparation are of sufficient chain length.
• Weight average molecular weight refers to the weight average in daltons of chains of uronic acid/hexosamine disaccharide repeats. The presence of non- uronic acid and/or non-hexosamine building blocks are not included in determining the weight average molecular weight. Thus, the molecular weight of non-uronic acid and non-hexosamine building blocks within a chain or chains in the preparation should not be included in determining the weight average molecular weight.
• the variable ci is the concentration of the polymer in slice i and Mi is the molecular weight of the polymer in slice i.
• the summations are taken over a chromatographic peak, which contains many slices of data. A slice of data can be pictured as a vertical line on a plot of chromatographic peak versus time. The elution peak can therefore be divided into many slices.
• the weight average molecular weight calculation is average dependant on the summation of all slices of the concentration and molecular weight.
• the weight average molar weight can be measured, e.g., using the Wyatt Astra software or any appropriate software.
• the weight average molecular weights described herein are determined by high liquid chromatography with two columns in series, for example a TSK G3000 SWXL and a G2000 SWXL, coupled with a multi angle light scattering (MALS) detector and a refractometric detector in series.
• the eluent used is a 0.2 sodium sulfate, pH 5.0, and a flow rate of 0.5 mL/min.
• chain length about 5 to 15 mole%, 7 to 14 mole%, or 9 to 12 mole% of the chains in a preparation can have ⁇ UHNA C ,6 S GHN S ,3 S ,6 S at, or within about two, four or six monosaccharides from the non-reducing end of the chain.
• Methods that can be used to quantify this structure include, e.g., capillary electrophoresis (CE) and high performance liquid chromatography (HPLC), e.g., reverse phase high performance liquid chromatography (RPHPLC).
• a response factor (RF) for ⁇ UH N Ac,6sGH N s,3S,6S can be determined.
• the determination can also include determining the RF for all species obtained, e.g., using CE or HPLC, e.g., a CE method described herein.
• a CE method described herein To obtain the RF for a species or all species obtained by CE, e.g., a CE method described herein, known concentrations of a standard for the specie or one or more of the species can be injected on the CE and used to determine a RF for each. The RF can then be used to determine the mole %.
• the sample has an actual level of a structure, which can be expressed, e.g., as 5 to 15 when described in units of mole %. That actual level can also be expressed in other units, e.g., weight %. That actual level is the same regardless of the units in which it is expressed.
• the specification of mole % in the method is merely to indicate the actual prevalence of the structure.
• the level of structure can be measured in terms of other units and the reference value can be expressed in terms of other units, as long as the reference value as expressed in terms of alternative units corresponds to the same amount of structure as the reference value expressed in mole %, 5 to 15 mole % in this example.
• a method which requires showing the structure is present at 5 to 15 mole % can be performed by showing that the structure is present in a range expressed in an alternative unit of measure, e.g., weight %, chain number, or %AUC, wherein the range, as described in the alternative unit of measure, corresponds to the same amount of the structure which would give the mole % referred to, in this example 5 to 15 mole %.
• an alternative unit of measure e.g., weight %, chain number, or %AUC
• a LMWH preparation described herein can have a mixture of ⁇ U and iduronic acid (I)/glucuronic acid (G) at the non-reducing end of the chains in the preparation.
• ⁇ U refers to an unsaturated uronic acid (iduronic acid (I), glucuronic acid (G) or galacturonic acid) that has a double bond introduced at the 4-5 position as a result, e.g., of the lyase action of a heparinase, a HSGAG lyase, or other enzyme having similar substrate specificity.
• the total number of chains in the preparation have a ⁇ U at the non-reducing end of the chain.
• the quantity of ⁇ U and/or I/G at the non-reducing end of chains within the sample can be determined using, e.g., 2D-NMR.
• the total number of chains having an acetylated hexosamine (H NAC ) at the reducing end and/or the number of open ring confirmations at the reducing end can be used to determine the total number of chains within the preparation.
• H NAC acetylated hexosamine
• the total percentage of chains having a ⁇ U and/or I/G at the non-reducing end can be compared to the total number of chains in the preparation.
• a LMWH preparation provided herein has substantially no modified reducing end structures. In preferred embodiments at least 85%, 90%, 95%, 98%, 99% or all of the chains in the LMWH preparation have a non-modified reducing end structure.
• a "modified reducing end structure” refers to a structure that arises at the reducing end of chains in the preparation due to the process of isolating or preparing the preparation from natural sources.
• many commercially available LMWH preparations are derived from unfractionated heparin primarily through chemical or enzymatic depolymerization of the polysaccharide chains.
• a process used to make a LMWH can cause one or more unique structural modifications to the reducing end of polysaccharide chains of starting material from a natural source.
• nitrous acid depolymerization of heparin results in the formation of a 2,5-anhydromannose at the reducing end, which can be reduced to form an alcohol
• depolymerization through esterification of the carboxylate functional group on the uronic acid followed by ⁇ - elimination results in the formation of a 1 ,6-anhydro structures at the reducing end of some chains.
• 2,5-anhydromannose and 1,6 anhydro structures are examples of modified reduce end structures that can be found on some chains of LMWHs.
• the chains in a LMWH preparation provided herein can include, e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99% or all of the chains having an acetylated hexosamine at the reducing end.
• Anti-Xa activity of a LMWH preparation plays a role in biological activity of LMWH preparations.
• a LMWH preparation provided herein has an anti-Xa activity of about 100 to 400 IU/mg, e.g., about 120 to 380 IU/mg, e.g., about 150 to 350 IU/mg, e.g., about 170 to 330 IU/mg, e.g., about 180 to 300 IU/mg, e.g., about 150 to 200 IU/mg, 200 to 300 IU/mg.
• Anti-Xa activity of a LMWH preparation is calculated in International Units of anti-factor Xa activity per milligram using the statistical methods for parallel line assays.
• the anti-factor Xa activity of LMWH preparations described herein is measured using the following principle:
• the anti-factor Xa activity is determined by the sample potentiating effect on antithrombin (ATIII) in the inhibition of activated Factor Xa (FXa). Factor Xa excess can be indirectly spectrophotometrically measured. Anti-factor Xa activity can be measured, e.g., on a Diagnostica Stago analyzer with the Stachrom® Heparin Test kit, on an ACL FuturaTM Coagulation system with the Coatest® Heparin Kit from Chromogenix, or on any equivalent system. Analyzer response can be calibrated using the NIBSC International Standard for Low Molecular Weight Heparin.
• LMWH preparations provided herein have an anti-Xa activity to anti-IIa activity ratio of 3:1 or less, e.g., 2.1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1 :1, 1 :1.
• Methods of determining anti-factor Xa activity and the anti-factor Ha activity have been described above.
• the ratio of anti-factor Xa activity to anti-factor Ha activity is calculated by dividing anti-factor Xa activity (dry basis) by the anti- factor Ha activity (dry basis).
• Both anti-Xa activity and anti-IIa activity of heparin and LMWH preparations involve binding of antithrombin III (ATIII) to a specific sequence, represented by the structure ⁇ UH N Ac,6sGH N s,3S,6S, within chains present in the preparation. Binding of ATIII to this sequence mediates anti-Xa activity.
• thrombin factor Ha
• thrombin factor Ha
• anti-IIa activity requires the presence of an ATIII binding site as well as a chain of sufficient length distal to the ATIII binding site.
• the anti-IIa activity of LMWH preparations provided herein can be attributed, at least in part, to the presence of ⁇ UH N Ac,6sGH N s,3S,6S at or near the non-reducing end of chains within the LMWH preparations as well as the length of many of the chains present in the preparation. This combination may result in chains within the preparation that contribute to both anti-Xa activity and anti-IIa activity.
• both anti-Xa activity and anti-IIa activity are provided by the same chain or chains, the clearance of that chain or chains can result in both a decrease in anti-Xa activity and anti-IIa activity. As such, the anti-Xa activity and anti-IIa activity can remain relatively constant over the course of administration.
• the LMWH preparations provided herein have an anti-Xa activity to anti-IIa activity remains relatively constant over the course of an administration of LMWH, e.g., the anti-Xa activity to anti-IIa activity ratio varies about ⁇ 1.5, ⁇ 1, ⁇ 0.5, or +0.2, over a period of about 30, 60, 120, 180, 240, 300 minutes.
• the ratio measured at a second time e.g., 30, 60, 120, 180, 240, 300 minutes
• the initial administration preferably be less than 3, and preferably at or around 2.
• LMWH preparations provided herein can be neutralized by protamine sulfate.
• anti-IIa activity and/or anti-Xa activity can be neutralized by at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% by administration of protamine.
• Protamine sulfate is commercially available, e.g., from Eli Lilly and Company.
• Neutralization of anti-Xa activity and anti-IIa activity can be measured, e.g., by standard coagulation assays such as ACT and aPTT, both of which are described further herein.
• Protamine sulfate can be administered intravenously, e.g., at a dose of about 1, 2, 3 mg per 100 anti-Xa IU of the LMWH preparation in plasma.
• protamine neutralization of anti-Xa activity and/or anti-IIa activity occurs within 5, 10,15, 20, 25, or 30 minutes after administration of the protamine sulfate.
• the polydispersity of LMWH preparations provided herein is about 1.6 or less, e.g., about 1.6 or 1.5 to 1.1, and numbers in between.
• polydisperse or “polydispersity” refers to the weight average molecular weight of a composition (Mw) divided by the number average molecular weight (Mn).
• the variable ci is the concentration of the polysaccharide in slice i
• Mi is the molecular weight of the polysaccharide in slice i.
• the summations are taken over a chromatographic peak, which contains many slices of data. A slice of data can be pictured as a vertical line on a plot of chromatographic peak versus time. The elution peak can therefore be divided into many slices.
• the number average molecular weight is a calculation dependent on the molecular weight and concentration at each slice of data. Methods of determining weight average molecular weight are described above, and were used to determine polydispersity as well.
• the ranges can be those ranges disclosed as well as other ranges.
• a range constructed from a lower endpoint of one range, e.g., for a given building block or activity can be combined with the upper endpoint of another range, e.g., for the given building block or activity, to give a range.
• LMWH preparation is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the LMWH is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
• substantially free means that a preparation of LMWH is at least 50% pure (wt/wt).
• the preparation of LMWH has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-heparin polysaccharides, proteins or chemical precursors or other chemicals, e.g., from manufacture. These also referred to herein as "contaminants”.
• contaminants that can be present in a LMWH preparation provided herein include, but are not limited to, calcium, sodium, heparinase enzyme (or other enzyme having similar substrate specificity), methanol, ethanol, chloride, sulfate, dermatan sulfate, and chondrotin sulfate.
• LMWH preparation The activity of a LMWH preparation provided herein can be monitored by standard anti-coagulation assays.
• assays include, e.g., ACT and aPTT, both of which are routinely practiced in hospitals and specifically hospital operating rooms.
• ACT is a test that is used to monitor the effectiveness of heparin therapy.
• the ACT can be done at the bedside, e.g., for patients experiencing pulmonary embolus, extracorporeal membrane oxygenation (ECMO) and hemodialysis.
• ACT is most often used before, during and after surgical intervention such as, e.g., cardiopulmonary bypass (CPB) surgery, PCI and stent placement.
• CPB cardiopulmonary bypass
• Reference value for the ACT can range from between 70-180 seconds. However, for certain procedures such as CPB the desired range can exceed 400-500 seconds.
• ACT utilizes negatively charged particles for a determination of time to clot formation.
• Examples of various particles that can be used include celite, which has a normal length of ACT being about 100 to 170 seconds; kaolin, which has a normal length of ACT being about 90 to 150 seconds; and glass particles, which have a normal length of ACT being about 190 to 300 seconds.
• Suitable machines for measuring ACT include, e.g., Hemochron and Medtronic HemoTec.
• a contact activator is used to stimulate the production of Factor XIIa by providing a surface for the function of high molecular weight kininogen, kallikrein and Factor XIIa. This contact activation is allowed to proceed for a specific period of time. Calcium is then added to trigger further reactions and the time required for clot formation is measured. Phospholipids are required to form complexes, which activate Factor X and Prothrombin.
• APTT can be measured by the IL TestTM APTT-SP(liquid). Reference values for aPTT is about 25 to 35 seconds. A prolonged aPTT indicates that clotting is taking longer than expected, e.g., due to a heparin or LMWH treatment.
• LMWH preparations e.g., a LMWH preparation described herein are also contemplated.
• methods include a method of making a LMWH preparation having an average chain length of about 8 to 16 or 9 to 16 disaccharides.
• the method includes providing a precursor LMWH preparation having a chain length of less than 8 to 16 or 9 to 16 disaccharides, and processing the precursor LMWH preparation to obtain a LMWH preparation having an average chain length of about 8 to 16 or 9 to 16 disaccharides.
• the precursor has an average chain length of about 8 to 14, e.g., 8 to 12, disaccharides.
• the precursor LMWH preparation can have the following structure:
• a precursor LMWH preparation used in this method can be obtained by a method that includes salt precipitation followed by (and) enzymatic digestion.
• a salt of a monovalent or divalent cation can be used in the method of obtaining the precursor LMWH preparation. Examples of monovalent and divalent cations that can be used include, e.g., sodium, potassium, rubidium, cesium, barium, calcium, magnesium, strontium, and combinations thereof.
• the salt can be, e.g., an acetate of a monovalent or divalent cation.
• Enzymatic digestion to obtain the LMWH precursor can include the use of one or more enzymes that cleaves at one or more glycosidic linkages of unsulfated uronic acids.
• Exemplary enzymes include heparinase III, mutants of heparinase III and HSGAG lyase III from B 'acteroides thetaiotaomicron.
• Heparinase III is described, for example, in U.S. Patent Nos: 5,681,733 and 5,919,693. Mutants of heparinase III are described in U.S. Patent No: 5,896,789.
• Preferred heparinase III mutants are those mutants having one or more histidine at His36, HislO5, Hisl 10, Hisl39, Hisl52, His225, His234, His424, His469 and His539 substituted with an alanine.
• the precursor LMWH preparation can be processed by size dependent separation such as, e.g., size exclusion chromatography, ion exchange chromatography and filtration. Further processing steps can be used prior to or after the size dependant separation, e.g., to obtain drug product.
• size dependent separation such as, e.g., size exclusion chromatography, ion exchange chromatography and filtration. Further processing steps can be used prior to or after the size dependant separation, e.g., to obtain drug product.
• drug product refers to a LMWH preparation having the purity required for and being formulated for pharmaceutical use.
• drug substance refers to a LMWH preparation having the polysaccharide constituents for pharmaceutical use but is not necessarily in its final formulation and/or comprises one or more non-product contaminant (e.g., one or more inorganic product such as sulfate, chloride, protein contaminant, process by-product such as heparinase, calcium, sodium).
• non-product contaminant e.g., one or more inorganic product such as sulfate, chloride, protein contaminant, process by-product such as heparinase, calcium, sodium.
• LMWH preparation includes providing a "fast moving fraction" from a glycosaminoglycan (GAG) containing sample, e.g., UFH .
• GAG glycosaminoglycan
• the fast moving fraction can be made as follows:
• a GAG containing sample e.g., UFH
• a first a precipitation e.g., with a polar organic solvent (e.g., an alcohol, e.g., ethanol), a polar non-organic solvent (e.g., water), and a salt (preferably, a sodium salt, e.g., sodium acetate), to yield a first supernatant;
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• a salt preferably, a sodium salt, e.g., sodium acetate
• a second precipitation e.g., with a polar organic solvent(e.g., an alcohol, e.g., ethanol), and a polar non-organic solvent (e.g., water), to yield a precipitate (this precipitate contains the fast moving fraction);
• a polar organic solvent e.g., an alcohol, e.g., ethanol
• a polar non-organic solvent e.g., water
• the fast moving fraction has the following structure:
• X is Na or Ca
• R is H or SO 3 Na
• This composition can occur as an intermediate in the production of a LMWH, e.g., as the product of precipitations to provide a fast moving fraction (as discussed herein).
• the fast moving fraction can be processed further to provide a LMWH of the invention.
• Processing of the fast moving fraction can include digesting the fast moving fraction with a chemical or enzyme that cleaves one or more glycosidic linkages of unsulfated uronic acid, e.g., one or more glycosidic linkages of unsulfated uronic acid adjacent to an N-acetyl glucosamine residue, e.g., to give rise to a preparation with the qualities and characteristics described herein.
• Enzymes can be evaluated for substrate specificity by the following steps: 1) functional screening of enzyme activity against two HSGAG substrates having different sulfation densities, e.g., heparin and heparan sulfate, whereby enzymes having a preference for heparan sulfate over heparin are selected; 2) fragment mapping of cleaved substrates from step 1 to assess substrate specificity; 3) cleavage of a LMWH such as Ml 18-REH step I intermediate or dalteparin using the enzyme, followed by; 4) assessment of anti-Xa activity and anti-IIa activity of the cleaved substrate using an in vitro assay; and 5) assessment of molecular weight distribution (or average chain length) of cleaved substrate using gel permeation chromatography (GPC) and/or size exclusion chromatography interfaced with multi- angle light scattering (SEC-MALS).
• GPC gel permeation chromatography
• SEC-MALS multi
• Step 1 assesses an enzyme's ability to act as an HSGAG lyase identified by the ability to generate an unsaturated C4-C5 bond at non-reducing ends of cleavage products as well as the enzymes preference for undersulfated substrates such as heparan sulfate.
• Enzyme activity can be followed spectrophotometrically by monitoring UV absorbance at 232 nm. An absorbance at this wavelength indicates formation of unsaturated uronic acids at the non-reducing ends of the cleavage product.
• Enzyme activity is monitored both kinetically (initial rate of product formation) and in terms of total product formation following exhaustive digestion (about 12 to 15 hours).
• Preferred enzymes have about a two fold preference for heparan sulfate over heparin and greater than a two fold (e.g., a 3 to 5 fold) difference in total activity.
• the second step assesses the cleavage specificity of the enzyme.
• Enzymes suitable for making the LMWH compositions described herein preferentially cleave undersulfated regions of heparin or heparan sulfate. IfUFH is the substrate used, this preference is demonstrated by an obvious underdigestion of substrate (as indicated by the presence of longer oligosaccharides) with any disaccharides being produced having a low sulfate density. In contrast when the substrate is heparan sulfate, digestion results in a greater number of disaccharides which indicates a higher cutting frequency.
• enzymes include heparinase III, mutants of heparinase III and HSGAG lyase from Bacteroides thetaiotaomicron.
• the fast moving fraction is processed, at least in part, with a mutated heparinase III having an alanine at residue 225 of the amino acid sequence of heparinase III instead of a histidine.
• This enzyme is also referred to herein as "MOH”.
• the digested LMWH preparation can be the final product, e.g., the drug substance or drug product, or can be further processed to obtain the final product, e.g., drug substance or drug product.
• the concentrated LMWH preparation can be further processed, e.g., by one or more of size dependant separation (e.g., by size exclusion chromatography, ion exchange chromatography and filtration), and filtration.
• size dependant separation e.g., by size exclusion chromatography, ion exchange chromatography and filtration
• the concentrated LMWH preparation is further processed by a size dependant separation, and the LMWH preparation obtained from this step has an average chain length of about 9 to 16 disaccharides.
• Analysis of a LMWH preparation such as an Ml 18-REH preparation using CE includes, e.g., digesting the preparation with one or more heparin degrading enzymes.
• the heparin degrading enzyme(s) can be, e.g., one or more heparinase, heparin lyase, HSGAG lyase, a lyase described as a GAG lyase that can also degrade heparin, and/or any polypeptide described as a hydrolase, sulfatase/sulfohyrdolase, or glycosyl hydrolase/glycosidase.
• the LMWH preparation can be digested with one or more of: an unsaturated glucuronyl hydrolase (e.g., F. heparinum ⁇ 4,5 glycuronidase, B. thetaiotaomicron ⁇ 4,5 glycuronidase); a glucuronyl hydrolase (e.g., mammalian ⁇ - iduronidase, ⁇ -glucuronidase); a sulfohydrolase (e.g., F. heparinum 2-O-sulfatase, 6-0- sulfatase, 3-O-sulfatase, B.
• an unsaturated glucuronyl hydrolase e.g., F. heparinum ⁇ 4,5 glycuronidase, B. thetaiotaomicron ⁇ 4,5 glycuronidase
• a glucuronyl hydrolase e.g., mammalian ⁇
• thetaiotaomicron 6-O-sulfatase a mucin desulfating enzyme, mammalian N-acetylglucosamine-6-sulfatase, mammalian iduronic acid-2-sulfatase); a N-sulfamidase (e.g., F.
• heparinum N-sulfamidase mammalian heparan-N-sulfatase
• an arylsulfatase e.g., a hexosaminidase; a glycosyl hydrolase (e.g., endo- N-acetyl glucosaminidase); a heparinase (e.g., Flavobacterum heparinum heparinase I, Flavobacterum heparinum heparinase II, Flavobacterum heparinum heparinase III, Flavobacterum heparinum heparinase IV); an endoglucoronidase (e.g., mammalian heparanase); a heparin/heparan sulfate lyase (e.g., Bacteroides thetaiotaomicron HSGAG lyase I
• a polypeptide described as above e.g., a heparinase or a heparin/heparin sulfate lyase
• a polypeptide described as above e.g., a heparinase or a heparin/heparin sulfate lyase
• Flavobacterium heparinum a.k.a. Pedobacter heparinus
• Haloarcula marismortui Agrobacterium tumefaciens, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus intermedius, Streptococcus suis, Enterococcus faecalis, Rhodopseudomonas palustris, Nitrobacter winogradskyi, Nitrobacter hamburgensis, Bradyrhizobium japonicum, Rhizobium meloliti, Mesorhizobium loti, Spinghobacterium sp., Brucella abortus biovar, Brucella melitensis, Solibacter usitatus, Acidobacterium capsulatum, Microbulbifer degradans, Pseudomonas aeruginosa, Burkholderia pseudomonascepacia, Geobacter metallireduc
• At least one enzyme used in the digestion is selected because it cleaves at specific linkages within heparins.
• the enzyme can be heparinase I and/or HSGAG lyase I.
• the LMWH preparation is digested with Flavobacterium heparinum heparinase I.
• the heparin preparation is digested with Bacteroides thetaiotaomicron HSGAG lyase I.
• enzymes can be selected for use in the digestion which resolve structures which could not be resolved solely with the use of heparinase I, II, and III. Any of the enzymes described herein can be replaced with an enzyme with functionally equivalent activity.
• the digestion is run to completion or at least sufficiently to provide a digest having all of the products found in Table 1OA and preferably substantially free of undigested material.
• the sample Prior to digestion, the sample can be lyophilized.
• the sample can be dried in a vacuum oven, e.g., at about 20 0 C, 25°C, 30 0 C, 35°C, 40 0 C, 43 0 C, 46 0 C, 49 0 C, 52 0 C, or 55 0 C, for about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.
• the sample can be lyophilized and/or dried under one of the following conditions:
• the sample can be lyophilized and/or dried under one of the following conditions: 40 0 C for 12 hours; 46°C for 8 hours; 49°C for 6 hours; 52°C for 4 hours.
• a sample can be suspended in water or a suitable buffer (e.g., 1 mM calcium acetate, 25 mM sodium acetate, pH 7.0, and 5% glycine) at a concentration of about 1, 2, 5, 10, 20, 50, 100, 200, or 500 mg/mL.
• a suitable buffer e.g., 1 mM calcium acetate, 25 mM sodium acetate, pH 7.0, and 5% glycine
• One or more heparin degrading enzyme can be added to the sample.
• heparinase I or HSGAG lyase I or combinations of these enzymes
• heparinase II or HSGAG lyase II or combinations of these enzymes
• heparinase III or HSGAG lyase III are added to the sample.
• the sample is digested at a temperature of about 18°C, 25°C, 30 0 C, 37°C, or 45°C for about 6, 12, 16, 18, 20 or 24 hours, e.g., at about 25°C for 24 hours; at 30 0 C for about 18 hours; at about 37°C for 12 hours.
• the enzyme or enzymes are removed from the sample mixture, e.g., using a Ni 2+ column, a size-exclusion column, dialysis, ultrafiltration, or the like.
• the enzyme or enzyme can be inactivated by heating (e.g., at 65 0 C for 20 minutes) following digestion.
• the sample can be stored, e.g., at -85 0 C, -70 0 C, -20 0 C, 4 0 C, 18 0 C, or 25 0 C for a period of time prior to analysis.
• Species separated by the methods described herein can be detected by numerous means, e.g., by ultraviolet absorbance (e.g., at a wavelength of about 232 nm), evaporative light scattering, fluorescence, pulsed amperometric detection, and mass spectrometry.
• ultraviolet absorbance e.g., at a wavelength of about 232 nm
• evaporative light scattering e.g., at a wavelength of about 232 nm
• fluorescence e.g., at a wavelength of about 232 nm
• mass spectrometry e.g., mass spectrometry.
• two or more means of detection can be utilized on the same sample, e.g., in series or in parallel.
• Additional enzyme digestions can be used to digest the sample.
• the digestion products are analyzed using an Agilent 3D Capillary Electrophoresis instrument.
• the capillary is an extended light path bare fused-silica capillary 75 ⁇ m ID, effective length 72 cm.
• Tris (5OmM), 10 ⁇ M dextran sulfate at pH 2.5 is used as CE buffer.
• Samples are injected at a pressure of 30 mbar for 20 seconds. Separation is performed at negative polarity and the analyte is monitored at 232 nm with 310 nm as the reference wavelength.
• New capillaries are pre-treated with a sequence of water, IN sodium hydroxide, water, and separation buffer. For each sample analysis, the capillary is preconditioned with buffer for 5 minutes.
• CE using e.g., an uncoated fused silica capillary, can be used to analyze LMWH, e.g., LMWH preparations described herein.
• LMWH e.g., LMWH preparations described herein.
• separation is dictated by analyte electrophoretic mobility almost exclusively. Due to the fact that all LMWH related saccharides have a net negative charge due to the carboxylate and sulfate moieties, separation is conducted under reverse polarity.
• supplementation of the low pH (pH2.5) buffer with dextran sulfate prevents non-specific absorption of anionic heparin-like material, enabling symmetrical peaks shapes and accurate quantification.
• the species in LMWH preparation can be resolved with a series of five digests (discussed in detail elsewhere herein); each digest is subjected to capillary electrophoresis after addition of an internal standard naphthalene monosulfonate.
• Mass recovery in the compositional analysis methodology was evaluated as follows. This analysis occurred at two levels: (1) mass recovery after enzymatic digestion and (2) mass recovery after separation with capillary electrophoresis.
• Two dimensional nuclear magnetic resonance spectroscopy (2D NMR) can be used as a means of partially resolving and identifying signals with minimum signal overlap. Integration of the 2D NMR signals followed by simple calculations can facilitate a quantitative monosaccharide compositional analysis of a polysaccharide mixture such as analysis of a LMWH preparation such as those provided herein. Moreover, 2D NMR can provide information on linkage environments of a disaccharide constituent, for example an H-U disaccharide, providing analysis of disaccharide linkages, including both qualitative and quantitative analysis.
• 2D NMR analysis can provide information about the epimerization state of a H-U linkage, for example, providing information as to whether the epimerization state is an iduronic acid residue or an glucuronic acid residue (i.e., I or U).
• a 2D proton-carbon correlation spectroscopy (HSQC) experiment can provide quantitative compositional analysis on one or more glycosaminoglycan.
• 2D NMR analysis can provide information about the nearest neighbor at the reducing end of a monosaccharide. This information can provide, for example, the sequence context in which a particular monosaccharide is present in a polysaccharide mixture, e.g., a LMWH such as a LMWH preparation described herein.
• 2D NMR method allows to discriminate between internal and reducing end residues.
• identification of measurable amounts of reducing N-acetyl glucosamine is peculiar of those LMWH described herein.
• 2D NMR analysis can provide information about the non- reducing end of LMWH chains, i.e. the amount of ⁇ UAp2-OH generated by the enzymatic digestion.
• 2D NMR can be used to evaluate a polysaccharide mixture for the presence of one or more impurities such as dermatan sulfate.
• impurities such as dermatan sulfate.
• the absence of a signal in the proton NMR at 2.06-2.09 ppm can be used to confirm that dermatan sulfate is not present at levels greater than the level of detection of the instrument (e.g., at a level greater than about 1%).
• saccharide structure is evaluated using, 2D NMR, e.g., e.g., sample of a polysaccharide mixture exchanged with D2O, lyophilized over night, and redissolved in D2O.
• the sample is then placed in an NMR tube for analysis and run at 303 K with a Bruker Avance 600 MHz spectrometer equipped with a 5 -mm TXI probe.
• Gradient-enhanced HSQC spectra is recorded with carbon decoupling during acquision.
• the data is then acquired with 16 scans for each of 256 increments in the indirect 13C dimension.
• the polarization transfer delay is set to 2.941 ms for an optimal transfer with IJCH scalar couplings of 155 Hz.
• the data is generally then processed, e.g. the matrix size IK x 256 is zero filled to 2K x IK by application of a squared-cosine function prior to Fourier transformation.
• Cross peaks are integrated, for example, using MestreC 4.5 software and only positive peaks are used for integration. Integrals are normalized to the H2/C2 peak of N-sulfated glucosamine (3.26 / 60.5 ppm). Peaks are generally assigned using published chemical shifts and experimental assignments via COSY and TOCSY experiments.
• Percent composition is calculated using the anomeric cross peak volumes, for which all uronic acid residues have similar IJCH couplings as do all glucosamine residues. For glucosamine anomeric peaks where overlapping prevents precise quantification, H2/C2 signals are integrated instead. The amount of every monosaccharide is expressed as percentage of the total glucosamine or uronic acid content. The ratio of 6-O-sulfation versus 6-O-desulfation is calculated from H6/C6 signal integration.
• the percent composition data is provided in Table 1 IA.
• compositions e.g., pharmaceutically acceptable compositions, which include a LMWH preparation described herein, formulated together with a pharmaceutically acceptable carrier, are provided.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• the carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).
• compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories.
• liquid solutions e.g., injectable and infusible solutions
• dispersions or suspensions e.g., dispersions or suspensions
• liposomes e.g., liposomes and suppositories.
• the preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions.
• the preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
• the LMWH preparation is administered by intravenous infusion or injection.
• the LMWH preparation is administered by intramuscular or subcutaneous injection.
• parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intra vitreous, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
• compositions typically should be sterile and stable under the conditions of manufacture and storage.
• the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high concentration.
• Sterile injectable solutions can be prepared by incorporating the active compound (i.e., LMWH in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, various polymers, monostearate salts and gelatin.
• the LMWH preparations can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
• the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
• a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, e.g., with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• a LMWH preparation provided herein can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
• the compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
• the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push- fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added.
• Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• the LMWH preparation may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• dry powder formations for inhalation therapy are within the scope of the invention. Such dry powder formulations may be
• compositions may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
• the compounds may also be formulated as a depot preparation.
• Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• compositions also may comprise suitable solid or gel phase carriers or excipients.
• suitable solid or gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
• compositions which can be used for non-parental delivery include: metered amounts of a composition to be administered from an inhaler for pulmonary delivery; tablets having a prescribed dosage unit for oral administration; transdermal patches to deliver a dosage unit across the skin; and suppositories to deliver a desired dosage unit rectally or vaginally.
• the compositions can be included in a container, pack, or dispenser together with instructions for administration.
• the LMWH preparation can also be administered with short or long term implantation devices.
• the preparation can be implanted subcutaneously, can be implanted into tissues or organs (e.g., the coronary artery, carotid artery, renal artery and other peripheral arteries, veins, kidney, heart cornea, vitreous, cerebrum, etc.), or can be implanted in physiological spaces around tissues and organs (e.g., kidney capsule, pericardium, thoracic or peritoneal space).
• tissues or organs e.g., the coronary artery, carotid artery, renal artery and other peripheral arteries, veins, kidney, heart cornea, vitreous, cerebrum, etc.
• physiological spaces around tissues and organs e.g., kidney capsule, pericardium, thoracic or peritoneal space.
• the LMWH preparation can also be used to coat various medical devices.
• the LMWH preparation can be used to coat a stent or extracorporeal circuit.
• Such formulations of the LMWH preparations may include using, e.g., controlled release beads, gel or microspheres as well as various polymers such as PLGA, cellulose, alginate or other polysaccharides.
• Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
• dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
• compositions of the invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of a LMWH preparation.
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
• a therapeutically effective amount of the LMWH preparation may vary according to factors such as the disease state, age, sex, and weight of the individual, and the LMWH preparation to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the LMWH preparation is outweighed by the therapeutically beneficial effects.
• a "therapeutically effective dosage” preferably inhibits a measurable parameter, e.g., coagulation or thrombosis, e.g., as measured by ACT and aPTT, by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
• a measurable parameter e.g., coagulation or thrombosis
• the ability of a compound to inhibit a measurable parameter, e.g., coagulation or thrombosis can be evaluated in an animal model system predictive of efficacy in humans. Alternatively, this property of a composition can be evaluated by examining the ability of the compound in an in vitro assay.
• Exemplary doses for intravenous administration of the LMWH preparation are about IU/kg to 200 IU/kg, e.g., 1 IU/kg; 2 IU/kg; 3 IU/kg, 4 IU/kg, 5 IU/kg, 6 IU/kg, 7 IU/kg, 8 IU/kg, 9 IU/kg, 10 IU/kg, 11 IU/kg, 12 IU/kg, 13 IU/kg, 14 IU/kg, 15 IU/kg, 16 IU/kg, 17 IU/kg, 18 IU/kg, 19 IU/kg, 20 IU/kg, 21 IU/kg, 22 IU/kg, 25 IU/mg, 30IU/kg, 40 IU/kg, 50IU/kg, 70 IU/kg, 100IU/kg, 125 IU/kg, 150IU/kg, 175 IU/kg, 200 IU/kg.
• exemplary doses for intravenous administration of the LMWH preparation are about 0.03 mg/kg to 0.45 mg/kg, e.g., 0.03 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.37 mg/kg, 0.4 mg/kg, 0.44 mg/kg, preferably aboutO.l mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.44 mg/kg, 0.47 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.7 mg/kg, preferably about 0.30 to 0.50 mg/kg, e.g., 0.30mg/kg, 0.35mg/kg, 0.40 mg/kg, 0.42 mg/kg, 0.44 mg/kg, 0.47 mg/kg or 0.50
• prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
• kits comprising a LMWH preparation provided herein.
• the kit can include one or more other elements including: instructions for use; other reagents, e.g., a therapeutic agent or protamine sulfate; devices or other materials for preparing the LMWH preparation for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
• Instructions for use can include instructions for monitoring anti-Xa activity and/or anti- Ha activity using coagulation assays such as ACT and aPTT.
• the instructions can include instructions for therapeutic application including suggested dosages and/or modes of administration, e.g., in a patient having a disorder, e.g., a disorder described herein.
• kits can further contain at least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agent as described herein, formulated as appropriate, in one or more separate pharmaceutical preparations.
• a diagnostic or therapeutic agent e.g., a diagnostic or therapeutic agent as described herein, formulated as appropriate, in one or more separate pharmaceutical preparations.
• the LMWH preparations can be used to treat a subject.
• the term "treat” or “treatment” is defined as the application or administration of a LMWH preparation to a subject, e.g., a patient, or application or administration to an isolated tissue or cell from a subject, e.g., a patient, which is returned to the patient.
• the subject can be a patient having a disorder (e.g., a disorder as described herein), a symptom of a disorder or a predisposition toward a disorder.
• the treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder.
• a subject is a vertebrate such as a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent.
• the subject can be, e.g., an experimental animal, a veterinary animal, or a human subject.
• a treatment can be therapeutic, e.g., a treatment which cures, heals, alleviates, relieves, alters, remedies, ameliorates, palliates, improves or affects the disorder or a symptom of the disorder, e.g., lessens, mitigates or ameliorates an existing unwanted condition or symptom thereof, or prophylactic, e.g., a treatment which delays, e.g., prevents, the onset of an unwanted condition or symptom thereof.
• LMWHs have many therapeutic utilities.
• the LMWH preparations provided herein can be used for the treatment of any type of condition in which heparin or LMWH therapy is useful.
• the preparations and methods are useful in a variety of in vitro, in vivo and ex vivo methods.
• heparins and LMWHs are useful for preventing and treating dementia, such as Alzheimer's disease, disorders associated with coagulation (e.g., DVT and PE), f ⁇ brotic disorders (e.g., major organ fibrosis, fibroproliferative disorders and scarring associated with trauma), thrombotic disorders (e.g., ACS, stable or unstable angina, MI (e.g., STEMI and NSTEMI)) or cardiovascular disease (atherosclerosis), vascular conditions or arterial fibrillation, allergy or respiratory disorders (e.g., asthma, emphysema, adult respiratory distress syndrome (ARDS), cystic fibrosis, and lung reperfusion injury), circulatory shock and related disorders, angiogenic disorders, cancer and metastatic disorders, sepsis, stenosis and restenosis, and osteoporosis.
• dementia such as Alzheimer's disease, disorders associated with coagulation (e.g., DVT and PE), f ⁇ brotic disorders (e.g., major organ
• the LMWH preparations provided herein can also be used on subjects having a fracture (e.g., a hip fracture) or to a subject prior to, during or after a surgical intervention (e.g., organ transplant, orthopedic surgery, hip replacement, knee replacement, PCI, stent placement, angioplasty and CABG).
• a fracture e.g., a hip fracture
• a surgical intervention e.g., organ transplant, orthopedic surgery, hip replacement, knee replacement, PCI, stent placement, angioplasty and CABG.
• organ transplant e.g., orthopedic surgery, hip replacement, knee replacement, PCI, stent placement, angioplasty and CABG.
• HLGAG compositions in various therapeutic methods is described and summarized in Huang, J. and Shimamura, A., Coagulation Disorders, 12, 1251-1281 (1998).
• the LMWH preparations are useful for treating or preventing disorders associated with coagulation.
• an imbalance in the coagulation pathway shifts towards excessive coagulation, the result is the development of thrombotic tendencies, which are often manifested as heart attacks, strokes, DVT, ACS, stable and unstable angina, and myocardial infarcts.
• a “disease associated with coagulation” as used herein refers to a condition characterized by local inflammation which can result from an interruption or reduction in the blood supply to a tissue which may occur, for instance, as a result of blockage of a blood vessel responsible for supplying blood to the tissue such as is seen for myocardial or cerebral infarction or peripheral vascular disease, or as a result of emboli formation associated with conditions such as arterial fibrillation, DVT or PE.
• DVT deep venous thrombosis
• DVT deep venous thrombosis
• This clotting occurs due to the absence of muscular activity in the lower extremities required to pump the venous blood (stasis), local vascular injury or a hypercoaguble state.
• the condition can be life -threatening if a blood clot migrates to the lung, resulting in a "pulmonary embolus” or otherwise interferes with cardiovascular circulation.
• One method of treatment involves administration of an anti-coagulant.
• Cardiovascular disease includes, but are not limited to, atherosclerosis and arterial fibrillation.
• Atrial fibrillation is a common form of arrhythmia generally arising as a result of emotional stress or following surgery, exercise, or acute alcoholic intoxication.
• Arterial fibrillation is characterized by disorganized arterial activity without discrete P waves on the surface ECG. This disorganized activity can lead to improper blood flow in the atrium and thrombus formation. These thrombi can embolize, resulting in cerebral ischemia and other disorders.
• Thrombotic disorders include, but are not limited to, ACS, e.g., MI, stable and unstable angina.
• Myocardial infarction is a disease state which sometimes occurs with an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by atherosclerosis. Such injury may be produced or facilitated by factors such as cigarette smoking, hypertension, and lipid accumulation.
• Angina is due to transient myocardial ischemia. This disorder is usually associated with a heaviness, pressure, squeezing, smothering, or choking feeling below the sternum. Episodes are usually caused by exertion or emotion, but can occur at rest.
• STEMI also referred to as "Q wave myocardial infarction” refers to MI with an abnormal echocardiogram.
• NSTEMI or “non-Q wave myocardial infarction” is not associated an echocardiogram abnormality.
• Stable angina occurs at predictable times with a specific amount of exertion or activity. Unstable angina may occur as a change in the usual pattern of stable angina. It my include chest pain that occurs at rest or with less and less exertion, that may be more severe and last longer, or that is less responsive to nitroglycerin. Unstable angina means that blood flow has gotten worse potentially by an increased narrowing or small blood clots that form in the coronary arteries. Unstable angina is a warning sign that myocardial infarction may soon occur.
• the LMWH preparation can be used for the treatment of thrombotic and cardiovascular disorders alone or in combination with other therapeutic agents for reducing the risk of a cardiovascular disease or for treating the cardiovascular disease.
• the combination therapy can include a LMWH preparation coformulated with, and/or coadministered with, one or more additional therapeutic agents, e.g., one or more therapeutic agent described herein.
• Administered "in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder or identified as at risk for the disorder and before the disorder has been prevented, cured or eliminated.
• the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap. This is sometimes referred to herein as “simultaneous" or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
• Other therapeutic agents include, but are not limited to, anti-inflammatory agents, anti-thrombotic agents, anti-platelet agents, fibrinolytic agents, thrombolytics, lipid reducing agents, direct thrombin inhibitors, anti-Xa inhibitors, anti-IIa inhibitors, glycoprotein Ilb/IIIa receptor inhibitors and direct thrombin inhibitors.
• GPIIb/IIIa inhibitors e.g., Integrelin
• P2Y12 inhibitors thienopyridine
• ticlopidine clopidogrel
• the monitorability by standard anticoagulation assays such as ACT and aPTT as well as the reversibility of the LMWH preparations provided herein provided improved flexibility in treating patients such as those patients admitted to the hospital and undergoing evaluation for possible cardiovascular surgery. Such benefits are highlighted by the following scenario.
• a patient goes the hospital complaining of symptoms that can be associated with various thrombotic disorders such as ACS including stable angina, unstable angina and MI.
• the monitorability and reversibility of the LMWH preparations provided herein allow use of such preparations while the patient is being evaluated for potential cardiovascular surgery.
• the monitorability of the LMWH preparations, the anti-Xa activity and anti-IIa activity of the LMWH preparation can be monitored during the procedure, and, if necessary, one or more additional doses of the LMWH preparation can be given during or after the procedure to maintain these activities. If it is determined that a patient will receive a surgical intervention such as CABG, the anti-Xa activity and anti-IIa activity of the LMWH preparation can be neutralized with protamine sulfate prior to surgical intervention. In addition, anti-Xa activity and anti-IIa activity can be monitored in the patient to ensure the activity is sufficiently decreased prior to the surgery.
• vascular conditions include, but are not limited to, disorders such as DVT, peripheral vascular disease, cerebral ischemia, including stroke, and PE.
• a cerebral ischemic attack or cerebral ischemia is a form of ischemic condition in which the blood supply to the brain is blocked. This interruption or reduction in the blood supply to the brain may result from a variety of causes, including an intrinsic blockage or occlusion of the blood vessel itself, a remotely originated source of occlusion, decreased perfusion pressure or increased blood viscosity resulting in inadequate cerebral blood flow, or a ruptured blood vessel in the subarachnoid space or intracerebral tissue.
• the methods are useful for treating cerebral ischemia.
• Cerebral ischemia may result in either transient or permanent deficits and the seriousness of the neurological damage in a patient who has experienced cerebral ischemia depends on the intensity and duration of the ischemic event.
• a transient ischemic attack is one in which the blood flow to the brain is interrupted only briefly and causes temporary neurological deficits, which often are clear in less than 24 hours.
• Symptoms of TIA include numbness or weakness of face or limbs, loss of the ability to speak clearly and/or to understand the speech of others, a loss of vision or dimness of vision, and a feeling of dizziness.
• Permanent cerebral ischemic attacks, also called stroke are caused by a longer interruption or reduction in blood flow to the brain resulting from either a thrombus or embolism.
• a stroke causes a loss of neurons typically resulting in a neurologic deficit that may improve but that does not entirely resolve.
• Thromboembolic stroke is due to the occlusion of an extracranial or intracranial blood vessel by a thrombus or embolus. Because it is often difficult to discern whether a stroke is caused by a thrombosis or an embolism, the term "thromboembolism" is used to cover strokes caused by either of these mechanisms.
• the methods are also directed to the treatment of acute thromboembolic stroke using a LMWH preparation provided herein.
• An acute stroke is a medical syndrome involving neurological injury resulting from an ischemic event, which is an interruption or reduction in the blood supply to the brain.
• An effective amount of a LMWH preparation alone or in combination with another therapeutic for the treatment of stroke is that amount sufficient to reduce in vivo brain injury resulting from the stroke.
• a reduction of brain injury is any prevention of injury to the brain which otherwise would have occurred in a subject experiencing a thromboembolic stroke absent the treatment described herein.
• physiological parameters may be used to assess reduction of brain injury, including smaller infarct size, improved regional cerebral blood flow, and decreased intracranial pressure, for example, as compared to pretreatment patient parameters, untreated stroke patients or stroke patients treated with thrombolytic agents alone.
• the LMWH preparation may be used alone or in combination with a therapeutic agent for treating a disease associated with coagulation.
• therapeutics useful in the treatment of diseases associated with coagulation include anticoagulation agents, antiplatelet agents, and thrombolytic agents.
• Anticoagulation agents prevent the coagulation of blood components and thus prevent clot formation.
• Anticoagulants include, but are not limited to, warfarin, Coumadin, dicumarol, phenprocoumon, acenocoumarol, ethyl biscoumacetate, and indandione derivatives.
• "Direct thrombin inhibitors" include hirudin, hirugen, Angiomax, agatroban, PPACK, thrombin aptamers.
• Antiplatelet agents inhibit platelet aggregation and are often used to prevent thromboembolic stroke in patients who have experienced a transient ischemic attack or stroke.
• Thrombolytic agents lyse clots which cause the thromboembolic stroke.
• Thrombolytic agents have been used in the treatment of acute venous thromboembolism and pulmonary emboli and are well known in the art (e.g. see Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S (1995); Holmes, et al, J Am Coll Cardiol; v.25 (7 suppl), p. 10S-17S(1995)).
• Pulmonary embolism refers to a disorder associated with the entrapment of a blood clot in the lumen of a pulmonary artery, causing severe respiratory dysfunction. Pulmonary emboli often originate in the veins of the lower extremities where clots form in the deep leg veins and then travel to lungs via the venous circulation. Thus, pulmonary embolism often arises as a complication of deep venous thrombosis in the lower extremity veins. Symptoms of pulmonary embolism include acute onset of shortness of breath, chest pain (worse with breathing), and rapid heart rate and respiratory rate. Some individuals may experience haemoptysis.
• Atherosclerosis is one form of arteriosclerosis that is believed to be the cause of most coronary artery disease, aortic aneurysm and atrial disease of the lower extremities, as well as contributing to cerebrovascular disease.
• the LMWH preparations are also useful before, during or after surgical and dialysis procedures. Surgical patients, especially those over the age of 40 years have an increased risk of developing DVT. Thus, the use of the LMWH preparations provided herein for preventing the development of thrombosis associated with surgical procedures is contemplated.
• general surgical procedures such as percutaneous intervention (e.g., percutaneous coronary intervention (PCI)), PCTA, stents and other similar approaches, hip or knee replacement, cardiac-pulmonary by-pass surgery, coronary revascularization surgery, orthopedic surgery, and prosthesis replacement surgery
• the methods are also useful in subjects undergoing a tissue or organ transplantation procedure or treatment for fractures such as hip fractures.
• the LMWH preparations provided herein are useful for treatment of respiratory diseases such as cystic fibrosis, asthma, allergy, emphysema, adult respiratory distress syndrome (ARDS), lung reperfusion injury, and ischemia-reperfusion injury of the lung.
• respiratory diseases such as cystic fibrosis, asthma, allergy, emphysema, adult respiratory distress syndrome (ARDS), lung reperfusion injury, and ischemia-reperfusion injury of the lung.
• Cystic fibrosis is a chronic progressive disease affecting the respiratory system.
• cystic fibrosis is Pseudomonas aeruginosa lung infection, which by itself accounts for almost 90% of the morbidity and mortality in cystic fibrosis.
• Therapeutics for treating cystic fibrosis include antimicrobials for treating the pathogenic infection.
• Asthma is a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively, associated with atopic or allergic symptoms. Asthma may also include exercise induced asthma, bronchoconstrictive response to bronchostimulants, delayed-type hypersensitivity, auto immune encephalomyelitis and related disorders. Allergies are generally caused by IgE antibody generation against allergens. Emphysema is a distention of the air spaces distal to the terminal bronchiole with destruction of alveolar septa. Emphysema arises out of elastase induced lung injury.
• Adult respiratory distress syndrome is a term which encompasses many acute defuse infiltrative lung lesions of diverse ideologies which are accompanied by severe atrial hypoxemia. One of the most frequent causes of ARDS is sepsis.
• Inflammatory diseases include but are not limited to autoimmune diseases and atopic disorders.
• Other types of inflammatory diseases which are treatable with the LMWH preparations provided herein are refractory ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune disease, non-specific ulcerative colitis, sepsis and interstitial cystitis.
• the LMWH preparations can be used to treat fibrotic disorders such as major organ fibrosis, fibroproliferative disorders and scarring associated with trauma.
• Major organ fibrosis includes, but is not limited to, interstitial lung disease (ILD), liver cirrhosis, kidney disease (e.g., diabetes and untreated hypertensive disease), heart disease and disorders of the eye (e.g., macular degeneration, retinal or vitreous retinopathy).
• ILD interstitial lung disease
• liver cirrhosis e.g., kidney disease (e.g., diabetes and untreated hypertensive disease), heart disease and disorders of the eye (e.g., macular degeneration, retinal or vitreous retinopathy).
• Fibroproliferative disorders include systemic and local scleroderma, keliods and hypertrophic scars, atherosclerosis, restenosis, fibrosarcoma and rheumatoid arthritis.
• scarring associated with trauma
• the LMWH preparations are used for inhibiting angiogenesis.
• Angiogenesis as used herein is the inappropriate formation of new blood vessels. "Angiogenesis" often occurs in tumors when endothelial cells secrete a group of growth factors that are mitogenic for endothelium causing the elongation and proliferation of endothelial cells which results in the generation of new blood vessels. Several of the angiogenic mitogens are heparin binding peptides which are related to endothelial cell growth factors. The inhibition of angiogenesis can cause tumor regression in animal models, suggesting a use as a therapeutic anticancer agent. Angiogenic disorders include, but are not limited to, neovascular disorders of the eye, osteoporosis, psoriasis, arthritis, cancer and cardiovascular disorders.
• the LMWH preparations may also be used inhibit cancer cell growth and metastasis.
• the methods are useful for treating and/or preventing tumor cell proliferation or metastasis in a subject.
• the cancer may be a malignant or non-malignant cancer.
• Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; leukemias, lymphomas; liver cancer; lung cancer (e.g.
• small cell and non small cell small cell and non small cell
• melanoma neuroblastomas
• oral cancer ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer, as well as other carcinomas and sarcomas.
• a subject in need of cancer treatment may be a subject who has a high probability of developing cancer.
• These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing a cancer and subjects exposed to cancer-causing agents such as tobacco, asbestos, or other chemical toxins, or a subject who has previously been treated for cancer and is in apparent remission.
• the LMWH preparation When administered to a patient undergoing cancer treatment, the LMWH preparation may be administered in cocktails containing other anti-cancer agents.
• the LMWH preparation may also be administered in cocktails containing agents that treat the side-effects of radiation therapy, such as anti-emetics, radiation protectants, etc.
• the treatments provided herein can further include administering protamine sulfate to neutralize the anti-Xa activity and/or anti-IIa activity of the LMWH preparation, e.g., once anti-coagulation or anti-thrombotic activity is no longer necessary.
• Protamine sulfate can be administered, e.g., by intravenous administration, at a dose of about 1, 2, 3 mg of protamine sulfate per 100 IU of anti-Xa activity.
• the IUs of anti-Xa activity can be determined using, e.g., the coagulation assays described herein.
• Step 1 of the process commercially available Unfractionated Heparin, USP (UFH) was subjected to a step-wise series of aqueous ethanol precipitations with calcium acetate in a 3:1 mass to mass ratio of calcium acetate to UFH to extract the portion of the UFH that is of lower molecular weight (also referred as the portion that is substantially the fast moving fraction).
• the resulting product of Step 1 fractionation was designated Intermediate 1.
• Step 2 involved the digestion of Intermediate 1 using a modified heparinase III enzyme having a substitution of an alanine for histidine at amino acid residue 225 (MOl 1) in aqueous sodium acetate buffer, pH 7.2 at 37 0 C to produce Intermediate 2.
• MOl 1 cleaved by ⁇ -elimination between N-acetylglucosamine residues and under sulfated uronic acids producing chains having a ⁇ 4,5 uronic acid group at the non-reducing end and an N-acetyl glucosamine at the reducing end.
• heat was turned off and sodium chloride was added to achieve a final solution concentration of approximately 2% w/v.
• Step 3 size exclusion chromatography (SEC) was used to separate the high anti- factor Xa and Ha components of Intermediate 2 away from the lower activity materials.
• the product of this step was designated Intermediate 3.
• Step 4 individual or combined Intermediate 3 materials were dissolved in purified water, filtered through a 0.2 pm filter, and lyophilized to produce Ml 18-REH drug substance.
• the starting material for making Ml 18-REH, UFH sodium (USP) is of porcine intestinal mucosa origin. In addition to the USP tests, additional controls have been put in place. These controls are listed in Table 1 :
• the Agarose Gel Electrophoresis semi-quantitatively separates the various components of heparin-based materials, as dermatan sulfate and chondroitan sulfate on the basis of their electrophoretic mobility.
• a horizontal separation in 0.5% agarose gel was conducted in barium acetate buffer pH 5.8, followed by 1,3-diaminopropane acetate pH 9 buffer.
• a special electrophoresis tank was employed, whereby the electrode chambers containing liquid buffer were overlaid with a water-immiscible, low density organic solvent (e.g., petroleum ether or heptane). This design provided efficient heat transfer between the agarose gel plate and a metal cooling tray filled with ice.
• Low molecular weight heparins traditionally are prepared from USP grade UFH.
• the UFH starting material for the Ml 18-REH process was restricted to those with potencies in excess of 160 IU/mg.
• Step 2 materials studied were not ideal substrates for the next step (enzymatic digestion) and attributes were identified that indicate preferred step 2 material.
• Table 2 and Table 3 below are data from 2D NMR analysis that illustrate the differences between the starting UFH and step 2 material. This analysis allows determination of the overall differences in structural attributes when going through Step 1 of the Ml 18-REH manufacturing process. Based on all the data obtained from the different analyses certain conclusions about the starting material, intermediates and manufacturing process were made.
• Table 2 2D NMR analysis of UFH and the step 2 materials obtained from them. These materials represent the preferred step 2 material.
• the step 2 materials had lower relative I 2 s content when compared to starting material and this was also reflected by the decrease in H NS -(I 2S ) structure as shown in the table. This was accompanied by a concomitant increase in the HN S -(I) and HN S -(G) structures as expected. Interestingly, there was also a relative increase in the amount of 6- O-sulfated hexosamine (H ⁇ s).
• Table 3 2D NMR analysis of UFH and the step 2 materials obtained from them. These materials represent the less preferred step 2 material.
• “Preferred” step 2 materials are those that are good substrates for the next step in the process i.e. enzymatic digestion by MOl 1, whereas "less preferred” step 2 materials are poorer substrates.
• H NAC internal
• the anti-factor Xa activity was measured as described herein.
• the anti- factor Xa activity was measured on either a Diagnostica Stago analyzer with the Stachrom® Heparin Test kit, or on an ACL FuturaTM Coagulation system with the Coatest® Heparin Kit from Chromogenix.
• the Analyzer response was calibrated using the NIBSC International Standard for Low Molecular Weight Heparin, lot 01/608 or current lot.
• the potency of Ml 18-REH Drug Substance was calculated in International Units of anti- factor Xa activity per milligram using the statistical methods for parallel line assays.
• the anti-factor Ha activity was measured as described herein, using the following principle.
• the anti-factor Ha activity was measured on either a Diagnostica Stago analyzer or on an ACL FuturaTM Coagulation system, with reagents from Chromogenix (S-2238 substrate, thrombin (53nkat/vial), and antithrombin).
• the Analyzer response was calibrated using the 2nd International Standard for Low Molecular Weight Heparin, lot 01/608 or equivalent.
• the potency of Ml 18-REH Drug Substance was calculated in International Units of anti-factor Ha activity per milligram using the statistical methods for parallel line assays.
• the weight average molar mass, the polydispersity and the molar mass distribution of Ml 18-REH Intermediates were measured using a Size Exclusion Chromatography (SEC) system attached to a Wyatt miniDAWN Multi Angle Light Scattering (MALS) detector or any other suitable MALS detector, and an Optilab rEX interferometric refractometer (RID) or other suitable RID in accordance with the USP ⁇ 621>, current version.
• SEC columns set consisted in columns packed with a high resolution L20 packing, for example a Tosoh SWXL guard column coupled with a Tosoh TSKgel G3000SWXL and a Tosoh TSKgel G2000SWXL in series.
• the system was equilibrated at 0.5 mL/min with a 0.2M sodium sulfate mobile phase whose pH was adjusted to 5.0 with sulfuric acid. Sodium azide was added at 0.05% in the mobile phase. The Ml 18-REH Intermediate was dissolved in the mobile phase to obtain a 10 mg/mL solution prior to injection.
• the weight average molar mass, the polydispersity and the distribution parameters were measured using the Wyatt Astra software or any appropriate software.
• the distribution was characterized by the percentage of chains with a molar mass lower than 5,500 Da (M5500), and the percentage of chains with a molar mass higher than 8,000 Da (Mgooo) or by the percentage of chains with a molar mass lower than 5,000 Da (M5500), and the percentage of chains with a molar mass higher than
• Step 2 of the Ml 18-REH process Intermediate 1 was digested with the MOl 1 enzyme. As the enzyme digested the Intermediate 1 substrate, it generated Intermediate 2 containing ⁇ 4,5 uronic acid residues possessing a characteristic UV232 absorbance. To monitor the progress of the digestion, the reaction solution was sampled periodically and the absorbance at 232 nm was measured. The Step 2 digestion was considered complete when the absorbance at 232 nm has not changed more than 2 AU in 1 hour.
• Table 5 shows the weight average molecular weight and distribution of various preparations of M 118-REH.
• Table 6 shows a comparison of M 118-REH, LMW and UFH products.
• composition analysis was performed with CE to identify and quantify individual building blocks that comprise the Ml 18-REH chains. These methods also identify the building block structure that is responsible for anti-Xa activity, referred to as "Anti-Xa Building Block”. Building Block Analysis / Compositional Analysis
• this method involves the enzymatic digestion of Ml 18-REH into its constituent building blocks followed by separation using CE ( Figures 2 A and 2B).
• CE is a high resolution separation technique and has been used extensively in the analysis of UFH and other glycosaminoglycans.
• the current method used capillary zone electrophoresis in an uncoated fused silica capillary. With capillary electrophoresis, the most highly sulfated species migrated through the capillary the fastest and are detected first.
• NMR spectroscopy has been successfully used for detecting and quantifying signals associated with major or minor structural features in polysaccharides. NMR spectroscopy is also one of the only techniques that allow an effective determination of the iduronic and glucuronic acid components in the mixture. Two dimensional (2D)NMR spectroscopy has been used as a means of resolving and identifying distinct signals that correspond to a certain population of monosaccharide residues. This approach enables one to not only quantify the basic monosaccharide constituents of the mixture, but to also assess their linkage environments in a quantitative manner.
• Two dimensional NMR provides a complementary technique to the compositional analysis of Ml 18-REH by CE.
• 2D NMR provides information on H-U linked disaccharides, thereby providing complementary analysis of disaccharide linkages.
• Recent methodology using 2D proton-carbon correlation spectroscopy (HSQC) experiments has demonstrated the ability to obtain this quantitative compositional analysis on glycosaminoglycans.
• Spectra of the anomeric region of Ml 18-REH, as measured using 2D proton- carbon correlation spectroscopy (HSQC) are presented in Figure 4. The cross peaks in the anomeric region are shown in the Figure.
• An analysis of the anomeric region of Ml 18-REH provided some very interesting information regarding what makes Ml 18-REH unique compared to other LMWHs.
• the anomeric region is much simpler when compared to other LMWHs, like enoxaparin.
• a key attribute of all LMWH products is that longer polysaccharide chains are cleaved into smaller fragments via a variety of depolymerization methods, as depicted in Figure 5. This cleavage event, which can be caused by either chemical or enzymatic reactions, results in characteristic signatures at both the reducing and non-reducing ends of the molecule.
• the characteristic end groups present is Ml 18-REH are described below.
• glucosamine structures at the new reducing ends on the Ml 18-REH chains were reflective of the specificity of the enzyme used in the process.
• the majority of the reducing end structures in Ml 18-REH were N-acetylated hexosamine (HNAC) residues.
• Ml 18-REH has a higher mole % of the antithrombin binding saccharide sequence as compared to the starting unfractionated heparin (UFH).
• Mi l 8-REH has negligible amount of linkage region as compared to the starting UFH.
• Ml 18-REH has a certain percentage on ⁇ 4,5 glucuronic acid at the non-reducing end of chains, whereas UFH does not contain this modified residue at the non- reducing end.
• Ml 18-REH is a heparin product having unique physical and functional attributes.
• the drug product, Ml 18-REH Injection is a clear, colorless to slightly yellow solution in a 3 rnL single use, Type 1 glass vial, sealed with a chlorobutyl stopper and oversealed with an aluminum crimp.
• Each vial nominally contains 5000 IU of anti-factor
• the quantitative composition of Ml 18-REH Injection is given in Table 11.
• the composition is given for the labeled volume of 2 mL.
• the vials were filled with 2.15 mL, consistent with the USP recommended excess volume.
• the amount of Ml 18-REH drug substance is calculated based on the anti-factor Xa activity (on a dried basis) and the Loss on Drying. Assuming anti-factor Xa activity of 200 lU/mg, the quantity of Ml 18- REH drug substance is 25 mg per vial.
• the quantity of Sodium Chloride required to achieve an osmolality of 280-330 mOsm/L is approximately 8 mg/mL, or 16 mg per vial.
• Ml 18-REH Injection was manufactured by dissolving Ml 18-REH Drug Substance in Water for Injection.
• Ml 18-REH Drug Substance is very soluble in aqueous solution and the particle size distribution of drug substance therefore had no effect on the performance of the drug product.
• Sodium Chloride USP was the only excipient used in Ml 18-REH Injection (at a concentration of approximately 8bg/mL). Sodium chloride was selected as an osmolality adjusting agent to avoid injection site discomfort and haemolysis upon administration.
• the Ml 18-REH Injection manufacturing process consisted of dissolving the Ml 18-REH drug substance in Water for Injection, USP, and adjusting the osmolality with Sodium chloride, USP.
• the formulated solution was filtered through two 0.2 pm filters in series and aseptically filled into vials. Heparin sodium products are subject to degradation at very high temperatures and therefore cannot be terminally sterilized.
• the process flow diagram for Ml 18-REH Injection is presented in Figure 6 (described below).
• FIG. 6 shows a process flow diagram for Ml 18-REH Injection.
• the amount of Ml 18-REH drug substance to be added to each batch was calculated based on the Assay (anti-factor Xa activity) and Loss on Drying values from the Certificate of Analysis according to the following calculation:
• Mi l 8-REH is the product of an enzymatic digest that results in a depolymerized low molecular weight heparin.
• the depolymerized pool is enriched for active anticoagulation and antithrombotic fractions, which may be a consequence of the site specific digestion by a specific glycosaminoglycan lyase.
• the specific site and orientation depolymerization via this enzyme has enabled Ml 18-REH to be a highly efficacious molecule on artery injury protection.
• Ml 18-REH has the attribute of being reversible and easily monitored by bedside clotting assays.
• Ml 18-REH In vitro anti-Xa activity of Ml 18-REH ranges from 180-300 IU/mg based on 2nd international low molecular weight heparin standards.
• Mi l 8-REH preparations higher in vitro anti-Xa/IIa activities are proportional to its fraction as ⁇ UHNac,6sGHNs,3S,6S containing 3-O-sulfation moiety.
• Ml 18-REH In vitro anti-IIa activity of Ml 18-REH ranges from 100-250 IU/mg based on 2 nd international low molecular weight heparin standards.
• Ml 18-REH can prolong the aPTT in vitro from 40 sec to 80 sec. at 2.4 ⁇ g/ml and aPTT change is propositional to the anti-Xa and anti-IIa activity.
• Protamine can fully reverse the anti-Xa activity of Ml 18-REH at ratio of 1 mg: 100 anti-Xa IU in human plasma. This can be compared to other LMWH preparations. For example, protamine can only neutralize 60% anti-Xa activity of enoxaparin at ratio of 3 mg: 100 anti-Xa IU in human plasma. These results are shown in Figure 7.
• HUVECs from ATCC were grown in 2% FBS F12K modified medium without ECGS.
• Ml 18-REH, Lovenox, and UFH were prepared in the same medium with the final concentration of 0.01 mg/ml and 0.005 mg/ml.
• Three wells of cells for each group were incubated under 37 0 C, 5% CO 2 , and 95% O 2 for 24 and 48 hours. The supernatant was taken out for TFPI release test using ELISA kit from ADI.
• Ml 18-REH has longer elimination half life than UFH and comparable to that of enoxaparin after intravenous injection.
• Ml 18-REH is quickly absorbed after subcutaneous injection with Tmax ranged from 1 hour to 3 hours.
• Ml 18-REH subcutaneous injection yields higher bioavailability in terms of both anti-Xa and anti-IIa activity than that of UFH, the bioavailability ranged from 50% to 100% compared with intravenous injection.
• the pharmacokinetics of Ml 18-REH is comparable to that of enoxaparin with elimination half life ranged from 3-5 hours and the major elimination mechanism is through renal excretion.
• NHP such as Cytomologus monkey
• the elimination half life of Ml 18-REH ranged from 20 minutes to 50 minutes.
• the anti-Xa/IIa ratio after intravenous injection was kept consistent during the PK course which ranged from 0.5 to 2 and Ml 18-REH was distributed in the circulation system and eliminated through renal route based on the analysis of pharmacokinetic parameters.
• Table 14 depicts pharmacokinetics of Ml 18- REH in NHP model.
• Anti-IIa 2 Mean 150 1.0 0.68 2.733 3.45 43.17 43.92
• the pharmacokinetics of Ml 18-REH represents the first order of elimination.
• ACT activated clotting time
• aPTT activated partial thromboplastin time
• activated clotting time presented dose response to Ml 18-REH escalating dosages.
• ACT increased 1.5-3 folds after Ml 18-REH delivery at 0.5 mg/kg while 2-4 folds at 1 mg/kg after intravenous injection.
• the pharmacodynamic profile was similar to that of the pharmacokinetics in terms of anti-Xa/IIa measurement; the elimination half life ranges from 0.15 to 0.5 hour.
• aPTT and ACT were measured after Ml 18-REH delivery intravenously.
• aPTT and ACT increased proportional to Ml 18-REH dose.
• the anti-Xa/IIa ratios were consistent after both intravenous and subcutaneous administration at 1.5 mg/kg.
• the anti- Xa/IIa ratios of both enoxaparin and UFH after intravenous administration fluctuated significantly during the course.
• Ml 18-REH intravenous bolus injection enhanced TFPI release into the blood stream by 2-20 folds and such effects last more than 24 hours.
• Ml 18-REH at a dose of 0.5 mg/kg significantly (p ⁇ 0.05) prolonged the time to occlusion (TTO) compared to saline (28.5 ⁇ 7.1 minutes versus 11. l ⁇ 0.9 minutes, respectively). Twenty- five percent (2/8) of the injured carotid artery injected with 0.5 mg/kg Ml 18-REH remained patent for the entire 60 minute observation period. In contrast, all (9/9) of the injured carotid arteries occluded in rats injected with saline within the 60 minute observation period.
• Ml 18-REH (lmg/kg) produced the greatest degree of protection from thrombosis in spite of lower anti-Xa activity than that at the 3 and 4 mg/kg doses of enoxaparin.
• the results are depicted in Figure 10 and Table 15.
• Ml 18-REH The antithrombotic and anticoagulant effects of Ml 18-REH were studied in a canine model of deep arterial thrombosis induced by severe electrolytic injury.
• IM intramuscular
• IM acepromazine 0.2 mg/kg, ⁇ 3 mg per animal
• a longitudinal incision was made on the medio-ventral surface of the neck to gain access to the tissues overlying the carotid arteries.
• One carotid artery and both jugular veins (one for backup) were subsequently exposed for approximately 2 cm by blunt dissection and supported by retaining ligatures at the proximal and distal ends.
• Two other incisions were made that initiated on the abdomen and extended distally along the pectineus for a distance covering 66%-75% of each femur.
• the fascia was opened at each incision, and the underlying femoral artery was exposed for a distance of approximately 2-3 cm.
• Twenty four anesthetized beagle dogs were instrumented with intravascular electrodes through the left and right femoral artery walls and positioned in direct contact with the intima. Each electrode was connected to a constant amperage power source, with the cathode placed at a distant subcutaneous site.
• a stenotic device was positioned immediately distal to the electrode, a pervascular Doppler flow probe was positioned proximal to the electrode, and a catheter was inserted into the carotid artery, all of which were connected to a Gould Ponemah Physiological Platform (Linton Instrumentation, Norfolk, UK) for continuous monitoring of arterial blood pressure, blood flow, and heart rate.
• a second catheter was inserted into the jugular vein for blood sample collection.
• an intravenous line was inserted into a peripheral vein for study treatment infusions, and limb leads were placed for electrocardiography (measured at Lead II).
• each animal received a continous IV infusion of vehicle (0.9% sterile saline) for 90 minutes.
• electrical current 300 ⁇ A was applied to the right femoral artery (control) through the intravascular electrode and administered continuously until full thrombus formation, defined as a reduction in Doppler flow to ⁇ 2% of baseline values, or until the end of the observation period at 180 minutes after initiation of electrolytic injury if the vessel remained patent.
• the vessel was ligated proximally and distally to the site of electrolytic injury, the segment was harvested, and any thrombus, if present, was weighed.
• the stenotic device was adjusted to limit hypoxia-induced reactive hyperemia to ⁇ 80% of the baseline response to physical occlusion (baseline reactive hyperemia was determined at each femoral artery immediately prior to vehicle or active-treatment infusions).
• baseline reactive hyperemia was determined at each femoral artery immediately prior to vehicle or active-treatment infusions.
• mean arterial pressure and heart rate were targeted to approximately 70 mm Hg and 100 beats per minute, respectively, via isoflurane anesthetic management.
• CBT cutaneous bleeding time
• O 2 saturation Assays for cutaneous bleeding time (CBT) and blood collections were carried out at protocol-specified time points (see below).
• ACT hematology and coagulation determinations
• whole blood samples (approximately 100 ⁇ L) were collected at 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, and 180 minutes for determination of ACT.
• ACT was assessed in a HEMOCHRON® Jr. Signature+ Microcoagulation System (ITC, Edison, NJ, USA) according to the manufacturer's instructions.
• Two aliquots (approximately 1.3 mL each) of whole blood were collected at 15 minutes, 60 minutes, and either 180 minutes or the time of occlusion, if applicable, for hematological and further coagulation assays.
• Hematologic parameters WBC, RBC, HGB, CHT, MCV, MCH, MCHC, PLT, RTC, ARTC, and WBC differentials
• WBC Hematologic parameters
• RBC Hematologic parameters
• Coagulation assays prothrombin time [PT], activated partial thromboplastin time [aPTT], and fibrinogen levels [FIB]
• PT prothrombin time
• aPTT activated partial thromboplastin time
• FIB fibrinogen levels
• ACT activated clotting time
• IU international unit
• SD standard deviation
• UFH unfractionated heparin
• Ml 18-REH at 150 anti-Xa IU/kg showed superior efficacy to UFH at 75 U/kg, despite the fact that the anticoagulant activity of Ml 18-REH and UFH were comparable at these doses.
• Mean times to occlusion were 59 ⁇ 25, 132 ⁇ 42, 165 ⁇ 23, 161 ⁇ 41, and 165 ⁇ 36 minutes in animals treated with vehicle, UFH, Ml 18-REH (37.5 anti-Xa IU/kg), Ml 18-REH (75 anti-Xa IU/kg), and Ml 18-REH (150 anti-Xa IU/kg), respectively. It should be noted that mean occlusion times in the active-treated arteries were underestimates of their true values, since many arteries, particularly in the Ml 18-REH treatment groups, did not fully occlude by the prespecif ⁇ ed endpoint of 180 minutes (such arteries were arbitrarily assigned an occlusion time of 180 minutes for the analysis).
• Mean thrombus weights were 24.0 ⁇ 9.15 mg in the vehicle-treated arteries, 19.4 ⁇ 9.47 mg in the UFH-treated arteries, and 24.5 ⁇ 12.17 mg, 19.8 ⁇ 6.24 mg, and 12.8 ⁇ 5.99 mg in the Ml 18-REH-treated arteries at 37.5, 75, and 150 anti-Xa IU/kg, respectively.
• the differences between treatment groups for mean time to occlusion and mean thrombus weight did not reach statistical significance.
• CBT varied from 80 ⁇ 15.5 to 160 ⁇ 52.5 seconds during vehicle administration at the protocol-specified time points in all groups (Table 17).
• UFH and Ml 18-REH treatment resulted in minimal increases in CBT, and the effects were highly variable.
• Group means ranged from 135 ⁇ 90.5 to 275 ⁇ 306.8 seconds after UFH treatment and 110 ⁇ 36.3 to 190 ⁇ 86.3 seconds after Ml 18-REH administration at all time points.
• Mean arterial pressure and heart rate during the course of the procedure in all treatment groups were 68-78 mm Hg and 96-111 bpm, respectively, close to the 70 mm Hg and 100 bpm targets pre-specified in the protocol.
• the maximum difference in mean arterial pressures and heart rate between the vehicle- and active -treated arteries in any given animal was 6 mm Hg and 5 beats per minute, respectively (P- values for differences were non- significant) .
• Ml 18-REH at 150 anti-Xa IU/kg showed statistically superior antithrombotic efficacy to a standard dose of UFH (75 U/kg), despite the fact that Ml 18-REH and UFH showed comparable activity in ACT, aPTT, and PT assays at these concentrations.
• UFH a standard dose of UFH
• Ml 18- REH was measurable in a dose-dependent fashion by point-of-care ACT assays. Its ACT response was well correlated to anti-Factor Xa activity compared with UFH. This feature may simplify administration of LMWHs in interventional and surgical settings.
• Ml 18-REH exhibited not only in vitro anti-Factor Xa activity, but also significant anti- Factor Ha activity, a characteristic that further distinguishes Ml 18-REH from currently available LMWH options (J. Hirsh et al.
• Heparin and low-molecular-weight heparin mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety
• Enoxaparin for instance, is characterized by a ratio of anti- Factor Xa activity to Ha activity of 17.2; by comparison, the analogous ratio for UFH is 3.3 (U. Cornelli and J. Fareed. "Human pharmacokinetics of low molecular weight heparins," Semin Thromb Hemost. 1999; 25 Suppl 3:57-61).
• Ml 18-REH had a ratio of anti-Factor Xa to Ha activity that was approximately 2-2.5 ( Figure 15), demonstrating enhanced anti-Factor Ha activity relative to other LMWHs.
• the ratio was generally more consistent over time than UFH, as predicted by the known variable metabolism of the large and polydisperse UFH molecules.
• Table 18 Summary Table of Thrombotic and Selected Hematologic Endpoints
• TTO Time to occlusion
• ACT Activated clotting time
• RFA Right femoral artery
• UFH unfractionated heparin
• Grp Group.
• Ml 18-REH protamine sulfate
• PS protamine sulfate
• Ml 18-REH was administered intravenously to conscious cynomologus monkeys, in some cases followed by administration of PS.
• Blood samples were obtained and evaluated for Ml 18- REH concentration as measured by anti-Xa and anti-IIa activity and coagulation profile. Hematology, cutaneous bleeding times and signs of clinical toxicity were monitored for 24 hours.
• Ml 18-REH activity was detectable immediately following iv administration at a dose of 150 anti-Xa IU/kg.
• First order elimination kinetics for Ml 18-REH was observed with tl/2 of 0.50 ⁇ 0.04 hr (anti-Xa) and 0.68 ⁇ 0.05 hr (anti-IIa).
• PS rapidly reversed Ml 18-REH anti-Xa and anti-IIa activity in a dose-dependent manner.
• the majority of anti-Xa (93.6 ⁇ 1.2%) and anti-IIa (90.14 ⁇ 1.2%) activity was rapidly neutralized by PS at a ratio of 1.5 mg PS per 100 IU anti-Xa activity, the higher dose studied.
• Ml 18-REH increased ACT 2-3 times and was reversed to baseline values within 5 minutes following intravenous PS administration. No signs of clinical toxicity or adverse bleeding were observed.
• Ml 18-REH causes a consistent and rapid anticoagulant affect that can be rapidly reversed by injection of PS.
• Ml 18-REH anticoagulation is easily measured and monitored by ACT and aPTT.
• the reversibility and monitorability of Ml 18-REH are unique compared with compared with other commercially available LMWHs.
• Ml 18-REH, enoxaparin and unfractionated heparin were administrated intravenously at different doses in rats or rabbits.
• Blood samples were obtained for ACT measurement and anti-Xa test.
• Hemachron Junior and ACT plus cuvette were used for ACT measurement while anti-Xa was measured with Coag-A-Mate MTX II.
• the correlation between anti-Xa and ACT was compared among these three heparins.
• 1 mg/kg Ml 18-REH and UFH were injected via ear marginal vein. Blood samples were collected at 5', 15', 30' 1, 2, 3, 4 hrs after heparin delivery.
• Ml 18-REH and UFH were dosed at 0.5 and 1 mg/kg while enoxaparin dosed at 1 mg/kg and 2 mg/kg. Those doses achieved significant anti-Xa and ACT elevation both in rats and rabbits.
• the correlation factor ( r 2 ) of anti-Xa to ACT was 0.79 in rabbits and 0.85 in rats.
• the correlation factors ( r 2 ) between anti-Xa and ACT for UFH were 0.31 in rabbits and 0.79 in rats, while for enoxaparin it was only 0.66 in rats. The results are show in Figure 12.
• Ml 18-REH presented first order of elimination with half life as 0.50 ⁇ 0.04 or 0.68 ⁇ 0.05 hour by anti-Xa and anti-IIa measurement, respectively.
• Distribution volumes (Vd) are 32.01 ⁇ 2.21 (anti-Xa) or 48.58 ⁇ 0.95 mL/kg (anti-IIa,) respectively.
• Clearance ( Cl ) are 37.24 ⁇ 4.65 (anti-Xa) and 43.92 ⁇ 5.38 (anti-IIa) mL/hr/kg.
• Ml 18-REH, enoxaparin and unfractionated heparin were administrated intravenously as a single bolus dose of 0.5 mg/kg via a marginal ear vein in rabbits.
• Bleeding time (BT) measurements were made on the ear at baseline and 5, 15, 30, 60, 120 and 180 minutes after test article administration.
• Ml 18-REH caused a 3-4 fold increase in BT at 5 minutes compared to 60 minutes post-administration (p ⁇ 0.05).
• a similar response in BT was observed by treatment with enoxaprin and UFH. Bleeding times returned to within normal range by 120 minutes and remained near baseline values for the remaining time points. No other adverse clinical findings were observed in any of treatment groups at the dose tested.
• Table 22 CBT of M118-REH after intravenous injection at 150 anti-Xa IU/kg in the NHP model.
• Half- lives for anti-Xa and anti-IIa activity in dog plasma ranged from 1.0 to 3.2 hours with no particular trend related to dosage or gender. The half-lives tended to be shorter on Day 13 than on Day 0. There was no consistent trend related to Ml 18 dosage with respect to the apparent systemic clearance or the apparent volume of distribution of anti-Xa and anti-IIa activities.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Health & Medical Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Medicinal Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Physical Education & Sports Medicine (AREA)
• Cardiology (AREA)
• Immunology (AREA)
• Heart & Thoracic Surgery (AREA)
• Pulmonology (AREA)
• Biomedical Technology (AREA)
• Rheumatology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Materials Engineering (AREA)
• Biochemistry (AREA)
• Hospice & Palliative Care (AREA)
• Polymers & Plastics (AREA)
• Oncology (AREA)
• Neurology (AREA)
• Neurosurgery (AREA)
• Molecular Biology (AREA)
• Vascular Medicine (AREA)
• Psychiatry (AREA)
• Hematology (AREA)
• Diabetes (AREA)
• Urology & Nephrology (AREA)
• Dermatology (AREA)
• Communicable Diseases (AREA)
• Transplantation (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Priority Applications (2)

Applications Claiming Priority (4)

Publications (1)

Family

ID=38722686

Family Applications (3)

Family Applications After (2)

Country Status (7)

Families Citing this family (50)

Family Cites Families (33)

• 2007
  • 2007-05-24 EP EP07797722A patent/EP2019843A2/de not_active Withdrawn
  • 2007-05-24 CA CA002652205A patent/CA2652205A1/en not_active Abandoned
  • 2007-05-24 WO PCT/US2007/069626 patent/WO2007140231A2/en active Application Filing
  • 2007-05-24 JP JP2009512295A patent/JP2009538386A/ja active Pending
  • 2007-05-24 AU AU2007267561A patent/AU2007267561B2/en not_active Ceased
  • 2007-05-24 CN CN2007800280960A patent/CN101495517B/zh not_active Expired - Fee Related
  • 2007-05-24 EP EP11172772A patent/EP2447285A3/de not_active Withdrawn
  • 2007-05-24 EP EP11172771A patent/EP2404939A3/de not_active Withdrawn
  • 2007-05-24 US US11/805,829 patent/US8609632B2/en active Active
• 2010
  • 2010-05-26 US US12/788,264 patent/US20100305022A1/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events